Oncostatin m receptor antigen binding proteins

ABSTRACT

The invention provides anti-oncostatin M receptor-β (OSMR) antigen binding proteins, e.g., antibodies and functional fragments, derivatives, muteins, and variants thereof. OSMR antigen binding proteins interfere with binding of OSM and/or IL-31 to OSMR. In some embodiments, anti-OSMR antigen binding proteins are useful tools in studying diseases and disorders associated with OSMR and are particularly useful in methods of treating diseases and disorders associated with OSMR and binding of OSM and/or IL-31 to OSMR.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalAppl. No. 61/829,082, filed May 30, 2013, the content of which isincorporated by reference in its entirety herein.

BACKGROUND OF THE INVENTION

Oncostatin M (OSM) and Interleukin-31 (IL-31) are members of the IL-6superfamily and share a receptor subunit, oncostatin M receptor-β (OSMR)(Dillon et al., Nat. Immunol. 5(7): 752-60, 2004). All of the members ofthis family, except IL-31, share the common chain of glycoprotein 130(gp130) in their multimeric receptor complexes. OSM signals through aheterodimeric receptor complex containing OSMR and gp130, while IL-31utilizes a gp130-like receptor, IL-31R, in combination with OSMR (Dillonet al., supra; Dreuw et al., J. Biol. Chem. 279(34): 36112-20, 2004). Ingeneral, OSMR and gp130 are expressed fairly ubiquitously across tissuesand cell types, and can be induced under a variety of stimulationconditions. IL-31R expression appears to be relatively more restrictedand tightly regulated. In human and mice alike, IL-31R mRNA expressionis detectable at low levels in tissues such as trachea, skeletal muscle,thymus and bone marrow (Dillon et al., supra). Although the level ofexpression is starkly different, both IL-31R and OSMR are co-expressedon a multitude of tissues, including skin and intestinal epithelialcells, suggesting those tissues should respond to IL-31 (Dillon et al.,supra; Dambacher et al., Gut 56(9): 1257-65, 2007). While OSMR isexpressed constitutively in the lung on epithelial cells, IL-31Rexpression is at negligible to low levels in the lung tissue, butupregulated upon various methods of airway challenge (Dillon et al.,supra; Jawa et al., J. Interferon Cytokine Res. 28(4): 207-19, 2008).

Secreted primarily by T lymphocytes, macrophages, and neutrophils, OSMand IL-31 are both upregulated in a variety of disease states thatinvolve inflammation. OSM has been implicated in diverse biologicalroles including bone formation, cartilage degradation, cholesteroluptake, pain and inflammation (Cawston et al., Arthritis Rheum.41(10):1760-71, 1998; Hasegawa et al., Rheumatology (Oxford) 38(7):612-7, 1999; Levy et al., J. Hepatol. 32(2): 218-26, 2000; Manicourt etal., Arthritis. Rheum. 43(2): 281-8, 2000; de Hooge et al., Am J.Pathol. 160(5):1733-43, 2002; Luzina et al., Arthritis Rheum 48(8):2262-74, 2003; Morikawa et al., J. Neurosci. 24(8): 1941-7, 2004; Konget al., J. Lipid Res. 46(6): 1163-71, 2005). OSM has been demonstratedto be a potent modulator of extracellular matrix (ECM) in a variety ofcontexts, suggesting that OSM is able to mediate seemingly oppositepathological consequences, including fibrosis (an excess of ECM) andcartilage degradation (a breakdown of ECM). Depending on tissue type andenvironmental milieu, both of these effects have been observed when OSMhas been overexpressed or exogenously administered into lungs or jointsof mice, respectively (Richards et al., Biochem. Soc. Trans. 30(2):107-11, 2002; Hui et al., Arthritis Rheum. 48(12): 3404-18, 2003; Rowanet al., Am. J. Pathol. 162(6): 1975-84, 2003). In addition, OSM haspreviously been shown to be upregulated in human pathologies where thesetypes of consequences exist (Cawston et al., supra; Hasegawa et al.,supra; Levy et al., supra; Manicourt et al., supra; Luzina et al.,supra). Predominantly, a locally-acting cytokine, OSM is upregulated inthe synovial fluid from joints of patients with rheumatoid arthritis(RA) (Cawston et al., supra; Manicourt et al., supra), in thebroncheoalevolar lavage (BAL) fluid of patients withscleroderma-associated interstitial lung disease (Luzina et al., supra),idiopathic pulmonary fibrosis (IPF), and in the livers of patients withcirrhosis (Levy et al., supra). The proposed impact on ECM by OSM can beattributed in part to the ability of OSM to shift the balance betweenmatrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs).TIMPs bind to MMPs in a 1:1 ratio with a high affinity that results in aloss of MMP proteolytic activity. TIMP-1 and TIMP-3 have been previouslyshown to be differentially regulated by OSM, resulting in an increase inTIMP-1 and a decrease in TIMP-3 (Gatsios et al., Eur. J. Biochem.241(1): 56-63, 1996). In addition to regulating the digestion ofextracellular matrix components, MMPs are also implicated in thecleaving and subsequent activation of a number of proteins, includingTGF-β, a potent pro-fibrotic cytokine (Leask et al., FASEB J. 18(7):816-27, 2004). OSM has also been reported to be capable of directlyinducing the transcription of type I collagen in vitro (Hasegawa et al.,J. Rheumatol. 25(2): 308-13, 1998).

Expression of both OSM and IL-31 has been found in the skin of patientswith psoriasis and atopic dermatitis, and mutations in OSMR and IL-31Rhave been linked to systemic cutaneous amyloidosis. System-widetransgenic overexpression of IL-31 induced a pruritic inflammatoryresponse in the skin of mice. Both OSM and IL-31 both signal throughOSMR on neurons where they have been suggested to promote nociceptiveand pruritic responses.

Collectively, these links to human diseases and the ability of OSM andIL-31 to promote a diverse array of pathologies, including at leastinflammation, extracellular matrix remodeling, pain, and pruritis,suggest blockade of OSMR is a useful target for therapeutic interventionin many diseases and disorders associated with OSMR.

SUMMARY OF THE INVENTION

The invention provides anti-OSMR antigen binding proteins, e.g.,antibodies and functional fragments thereof, having properties amenableto commercial production and therapeutic use in humans. The anti-OSMRantigen binding proteins are useful in methods of treating diseases anddisorders associated with OSMR and, particularly, those associated withthe binding of OSM or IL-31 to OSMR. Provided herein are OSMR-bindingantibodies that bind OSMR with high affinity and effectively block OSMand/or IL-31 binding to OSMR, thereby reducing OSMR-mediated signalingin the cell.

In a first aspect, the OSMR antigen binding protein comprises a) a lightchain variable domain having at least 90% identity, at least 95%identity, or is identical to the amino acid sequence set forth in SEQ IDNO:27, SEQ ID NO:28, or SEQ ID NO:29; b) a heavy chain variable domainhaving at least 90% identity, at least 95% identity, or is identical tothe amino acid sequence set forth in SEQ ID NO:9, SEQ ID NO:10, or SEQID NO:11; or c) the light chain variable domain of a) and the heavychain variable domain of b).

Preferred antigen binding proteins of the first aspect include thosecomprising a light chain variable domain having at least 90%, at least95%, or is identical to the amino acid sequence set forth in SEQ IDNO:27 and a heavy chain variable domain having at least 90%, at least95%, or is identical to the amino acid sequence set forth in SEQ IDNO:9; those comprising a light chain variable domain having at least90%, at least 95%, or is identical to the amino acid sequence set forthin SEQ ID NO:28 and a heavy chain variable domain having at least 90%,at least 95%, or is identical to the amino acid sequence set forth inSEQ ID NO:10; and those comprising a light chain variable domain havingat least 90%, at least 95%, or is identical to the amino acid sequenceset forth in SEQ ID NO:29 and a heavy chain variable domain having atleast 90%, at least 95%, or is identical to the amino acid sequence setforth in SEQ ID NO:11.

OSMR antigen binding proteins comprising a heavy chain variable domainhaving the above-defined sequence relatedness to SEQ ID NO:9 canoptionally contain an amino acid other than asparagine (for example,aspartic acid) at the position corresponding to position 73 in SEQ IDNO:9. In such embodiments, the heavy chain variable domain optionallycomprises the amino acid sequence set forth in SEQ ID NO:53.OSMR antigen binding proteins comprising a heavy chain variable domainhaving the above-defined sequence relatedness to SEQ ID NO:10 canoptionally contain an amino acid other than asparagine (for example,aspartic acid) at the position corresponding to position 73 in SEQ IDNO:10. In such embodiments, the heavy chain variable domain optionallycomprises the amino acid sequence set forth in SEQ ID NO:54.

In a second aspect, the OSMR antigen binding protein comprises a) alight chain variable domain having no more than ten or no more than fiveamino acid additions, deletions or substitutions from the amino acidsequence set forth in SEQ ID NO:27, SEQ ID NO:28, or SEQ ID NO:29; b) aheavy chain variable domain having no more than ten or no more than fiveamino acid additions, deletions or substitutions from the amino acidsequence set forth in SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11; or c)the light chain variable domain of a) and the heavy chain variabledomain of b).

Preferred antigen binding proteins of the second aspect include thosecomprising a light chain variable domain having no more than ten or nomore than five amino acid additions, deletions or substitutions from theamino acid sequence set forth in SEQ ID NO:27 and a heavy chain variabledomain having no more than ten or no more than five amino acidadditions, deletions or substitutions from the amino acid sequence setforth in SEQ ID NO:9; those comprising a light chain variable domainhaving no more than ten or no more than five amino acid additions,deletions or substitutions from the amino acid sequence set forth in SEQID NO:28 and a heavy chain variable domain having no more than ten or nomore than five amino acid additions, deletions or substitutions from theamino acid sequence set forth in SEQ ID NO:10; and those comprising alight chain variable domain having no more than ten or no more than fiveamino acid additions, deletions or substitutions from the amino acidsequence set forth in SEQ ID NO:29 and a heavy chain variable domainhaving no more than ten or no more than five amino acid additions,deletions or substitutions from the amino acid sequence set forth in SEQID NO:11.

OSMR antigen binding proteins comprising a heavy chain variable domainhaving the above-defined sequence relatedness to SEQ ID NO:9 canoptionally contain an amino acid other than asparagine (for example,aspartic acid) at the position corresponding to position 73 in SEQ IDNO:9. In such embodiments, the heavy chain variable domain optionallycomprises the amino acid sequence set forth in SEQ ID NO:53.OSMR antigen binding proteins comprising a heavy chain variable domainhaving the above-defined sequence relatedness to SEQ ID NO:10 canoptionally contain an amino acid other than asparagine (for example,aspartic acid) at the position corresponding to position 73 in SEQ IDNO:10. In such embodiments, the heavy chain variable domain optionallycomprises the amino acid sequence set forth in SEQ ID NO:54.

In a third aspect, the OSMR antigen binding protein contains a lightchain variable domain comprising a) an LCDR1 having no more than threeamino acid additions, deletions, or substitutions from the LCDR1sequence set forth in SEQ ID NO:30; an LCDR2 having no more than threeamino acid additions, deletions, or substitutions from the LCDR2sequence set forth in SEQ ID NO:33; and an LCDR3 having no more thanthree amino acid additions, deletions, or substitutions from the LCDR3sequence set forth in SEQ ID NO:36; b) an LCDR1 having no more thanthree amino acid additions, deletions, or substitutions from the LCDR1sequence set forth in SEQ ID NO:31; an LCDR2 having no more than threeamino acid additions, deletions, or substitutions from the LCDR2sequence set forth in SEQ ID NO:34; and an LCDR3 having no more thanthree amino acid additions, deletions, or substitutions from the LCDR3sequence set forth in SEQ ID NO:37; or c) an LCDR1 having no more thanthree amino acid additions, deletions, or substitutions from the LCDR1sequence set forth in SEQ ID NO:32; an LCDR2 having no more than threeamino acid additions, deletions, or substitutions from the LCDR2sequence set forth in SEQ ID NO:35; and an LCDR3 having no more thanthree amino acid additions, deletions, or substitutions from the LCDR3sequence set forth in SEQ ID NO:38; and a heavy chain variable domaincomprising d) an HCDR1 having no more than three amino acid additions,deletions, or substitutions from the HCDR1 sequence set forth in SEQ IDNO:12; an HCDR2 having no more than three amino acid additions,deletions, or substitutions from the HCDR2 sequence set forth in SEQ IDNO:15; and an HCDR3 having no more than three amino acid additions,deletions, or substitutions from the HCDR3 sequence set forth in SEQ IDNO:18; e) an HCDR1 having no more than three amino acid additions,deletions, or substitutions from the HCDR1 sequence set forth in SEQ IDNO:13; an HCDR2 having no more than three amino acid additions,deletions, or substitutions from the HCDR2 sequence set forth in SEQ IDNO:16; and an HCDR3 having no more than three amino acid additions,deletions, or substitutions from the HCDR3 sequence set forth in SEQ IDNO:19; or f) an HCDR1 having no more than three amino acid additions,deletions, or substitutions from the HCDR1 sequence set forth in SEQ IDNO:14; an HCDR2 having no more than three amino acid additions,deletions, or substitutions from the HCDR2 sequence set forth in SEQ IDNO:17; and an HCDR3 having no more than three amino acid additions,deletions, or substitutions from the HCDR3 sequence set forth in SEQ IDNO:20.

Preferred OSMR antigen binding proteins of third aspect include thosecomprising the light chain variable domain of a) and the heavy chainvariable domain of d); those comprising the light chain variable domainof b) and the heavy chain variable domain of e); and those comprisingthe light chain variable domain of c) and the heavy chain variabledomain of f).

OSMR antigen binding proteins comprising the light chain variable domainof a) and the heavy chain variable domain of d) can optionally contain aheavy chain variable domain that comprises an amino acid other thanasparagine (for example, aspartic acid) at the position corresponding toposition 73 in SEQ ID NO:9. In such embodiments, the heavy chainvariable domain optionally comprises the amino acid sequence set forthin SEQ ID NO:53.OSMR antigen binding proteins comprising the light chain variable domainof b) and the heavy chain variable domain of e) can optionally contain aheavy chain variable domain that comprises an amino acid other thanasparagine (for example, aspartic acid) at the position corresponding toposition 73 in SEQ ID NO:10. In such embodiments, the heavy chainvariable domain optionally comprises the amino acid sequence set forthin SEQ ID NO:54.

In a fourth aspect of the invention, the OSMR antigen binding protein ofthe first, second, or third aspect binds to human OSMR with an affinityof less than or equal to 1×10⁻¹⁰ M.

In a fifth aspect of the invention, the OSMR antigen binding protein ofthe first, second, third, or fourth aspect inhibits binding of human OSMto human OSMR and/or human IL-31 to human OSMR.

In a sixth aspect of the invention, the OSMR antigen binding protein ofthe first, second, third, fourth, or fifth aspect reduces humanOSM-mediated and/or human IL-31-mediated OSMR signaling in humanOSMR-expressing cells.

In a seventh aspect of the invention, the OSMR antigen binding proteinof the sixth aspect reduces cynomolgus monkey OSM-mediated and/orIL-31-mediated OSMR signaling in cynomolgus monkey OSMR-expressingcells.

In an eighth aspect of the invention, the OSMR antigen binding proteinof the first, second, third, fourth, fifth, sixth or seventh aspect isan antibody, such as a human antibody. Preferred antibodies includethose antibodies that comprise a light chain having the amino acidsequence set forth in SEQ ID NO:24 and a heavy chain having the aminoacid sequence set forth in SEQ ID NO:6; those that comprise a lightchain having the amino acid sequence set forth in SEQ ID:25 and a heavychain having the amino acid sequence set forth in SEQ ID NO:7; and thosethat comprise a light chain having the amino acid sequence set forth inSEQ ID:26 and a heavy chain having the amino acid sequence set forth inSEQ ID NO:8.

Additional antibodies include those antibodies that comprise a lightchain having the amino acid sequence set forth in SEQ ID NO:24 and aheavy chain having the amino acid sequence set forth in SEQ ID NO:50;those that comprise a light chain having the amino acid sequence setforth in SEQ ID:25 and a heavy chain having the amino acid sequence setforth in SEQ ID NO:51; and those that comprise a light chain having theamino acid sequence set forth in SEQ ID:26 and a heavy chain having theamino acid sequence set forth in SEQ ID NO:52.

In a ninth aspect, the invention provides nucleic acids or isolatednucleic acids encoding one or more polypeptide components of a OSMRantigen binding protein, e.g., an antibody light chain or antibody heavychain. In preferred embodiments the nucleic acid encodes a polypeptidecomprising:

a) a light chain variable domain having at least 95% identity to theamino acid sequence set forth in SEQ ID NO:27, SEQ ID NO:28, or SEQ IDNO:29;

b) a heavy chain variable domain having at least 95% identity to theamino acid sequence set forth in SEQ ID NO:9, SEQ ID NO:10, or SEQ IDNO:11;

c) a light chain variable domain having no more than five amino acidadditions, deletions or substitutions from the amino acid sequence setforth in SEQ ID NO:27, SEQ ID NO:28, or SEQ ID NO:29;

d) a heavy chain variable domain having no more than five amino acidadditions, deletions or substitutions from the amino acid sequence setforth in SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11;

e) a light chain variable domain comprising:

-   -   i) an LCDR1 having no more than three amino acid additions,        deletions, or substitutions from the LCDR1 sequence set forth in        SEQ ID NO:30; an LCDR2 having no more than three amino acid        additions, deletions, or substitutions from the LCDR2 sequence        set forth in SEQ ID NO:33; and an LCDR3 having no more than        three amino acid additions, deletions, or substitutions from the        LCDR3 sequence set forth in SEQ ID NO:36;    -   ii) an LCDR1 having no more than three amino acid additions,        deletions, or substitutions from the LCDR1 sequence set forth in        SEQ ID NO:31; an LCDR2 having no more than three amino acid        additions, deletions, or substitutions from the LCDR2 sequence        set forth in SEQ ID NO:34; and an LCDR3 having no more than        three amino acid additions, deletions, or substitutions from the        LCDR3 sequence set forth in SEQ ID NO:37; or    -   iii) an LCDR1 having no more than three amino acid additions,        deletions, or substitutions from the LCDR1 sequence set forth in        SEQ ID NO:32; an LCDR2 having no more than three amino acid        additions, deletions, or substitutions from the LCDR2 sequence        set forth in SEQ ID NO:35; and an LCDR3 having no more than        three amino acid additions, deletions, or substitutions from the        LCDR3 sequence set forth in SEQ ID NO:38; or

f) a heavy chain variable domain comprising:

-   -   i) an HCDR1 having no more than three amino acid additions,        deletions, or substitutions from the HCDR1 sequence set forth in        SEQ ID NO:12; an HCDR2 having no more than three amino acid        additions, deletions, or substitutions from the HCDR2 sequence        set forth in SEQ ID NO:15; and an HCDR3 having no more than        three amino acid additions, deletions, or substitutions from the        HCDR3 sequence set forth in SEQ ID NO:18;    -   ii) an HCDR1 having no more than three amino acid additions,        deletions, or substitutions from the HCDR1 sequence set forth in        SEQ ID NO:13; an HCDR2 having no more than three amino acid        additions, deletions, or substitutions from the HCDR2 sequence        set forth in SEQ ID NO:16; and an HCDR3 having no more than        three amino acid additions, deletions, or substitutions from the        HCDR3 sequence set forth in SEQ ID NO:19; or    -   iii) an HCDR1 having no more than three amino acid additions,        deletions, or substitutions from the HCDR1 sequence set forth in        SEQ ID NO:14; an HCDR2 having no more than three amino acid        additions, deletions, or substitutions from the HCDR2 sequence        set forth in SEQ ID NO:17; and an HCDR3 having no more than        three amino acid additions, deletions, or substitutions from the        HCDR3 sequence set forth in SEQ ID NO:20.        In certain embodiments, the nucleic acid or isolated nucleic        acid encodes a polypeptide that comprises the amino acid        sequence set forth in SEQ ID NO:53 or SEQ ID NO:54.        In certain embodiments, the nucleic acid or isolated nucleic        acid encodes a polypeptide that comprises the amino acid        sequence set forth in SEQ ID NO:50, SEQ ID NO:51, or SEQ ID        NO:52.

In certain embodiments of the ninth aspect, the nucleic acid or isolatednucleic acid encodes an antibody light chain and is at least 80%, atleast 90%, at least 95%, or is 100% identical to the nucleotide sequenceset forth in SEQ ID NO:21, SEQ ID NO:22, or SEQ ID NO:23. In otherembodiments of the ninth aspect, the nucleic acid or isolated nucleicacid encodes an antibody heavy chain and is at least 80%, at least 90%,at least 95%, or is 100% identical to the nucleotide sequence set forthin SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5.

In certain embodiments, the heavy chain is encoded by a nucleic acidcomprising a nucleotide sequence set forth in SEQ ID NO:47, SEQ IDNO:48, or SEQ ID NO:49.

In a tenth aspect, the invention provides an expression vectorcomprising one or more nucleic acids or isolated nucleic acids of theeighth aspect. In certain embodiments, the expression vector encodes anantibody light chain, an antibody heavy chain, or both an antibody lightchain and a heavy chain.

In an eleventh aspect, the invention provides a recombinant host cellcomprising one or more nucleic acids or isolated nucleic acids of theninth aspect operably linked to a promoter, including recombinant hostcells comprising one or more expression vectors of the tenth aspect ofthe invention. In preferred embodiments, the recombinant host cellsecretes an antibody that binds OSMR. Preferred host cells are mammalianhost cells, including CHO cell lines.

In a twelfth aspect, the invention provides methods of treating anautoimmune disorder, an inflammatory disorder, or a disorder associatedwith extracellular matrix deposition or remodeling, said methodcomprising administering a therapeutically effective amount of an OSMRantigen binding protein of any one of the first, second, third, fourth,fifth, sixth, sixth, seventh, or eighth aspects to a patient in needthereof. In preferred embodiments, the OSMR antigen binding protein isan antibody comprising a light chain variable domain amino acid sequenceas set forth in SEQ ID NO:27 and a heavy chain variable domain aminoacid sequence as set forth in SEQ ID NO:9 (e.g., Ab1), an antibodycomprising a light chain variable domain amino acid sequence as setforth in SEQ ID NO:28 and a heavy chain variable domain amino acidsequence as set forth in SEQ ID NO:10 (e.g., Ab2), or an antibodycomprising a light chain variable domain amino acid sequence as setforth in SEQ ID NO:29 and a heavy chain variable domain amino acidsequence as set forth in SEQ ID NO:11 (e.g., Ab3). In some embodiments,the OSMR antigen binding protein is an antibody comprising a light chainvariable domain amino acid sequence as set forth in SEQ ID NO:27 and aheavy chain variable domain amino acid sequence as set forth in SEQ IDNO:53, or an antibody comprising a light chain variable domain aminoacid sequence as set forth in SEQ ID NO:28 and a heavy chain variabledomain amino acid sequence as set forth in SEQ ID NO:54. In preferredembodiments, the OSMR antigen binding protein inhibits binding of OSM toOSMR or IL-31 to OSMR. In particularly preferred embodiments, theautoimmune disorder, inflammatory disorder, or disorder associated withextracellular matrix deposition or remodeling is fibrosis, cartilagedegradation, arthritis, rheumatoid arthritis, scleroderma,scleroderma-associated interstitial lung disease, idiopathic pulmonaryfibrosis, cirrhosis, psoriasis, atopic dermatitis, systemic cutaneousamyloidosis, primary cutaneous amyloidosis, inflammation, pruriticinflammation, prurigo nodularis, and pain.

In a thirteenth aspect, the invention provides a method of making anOSMR antigen binding protein of any one of the first, second, third,fourth, fifth, sixth, sixth, seventh, or eighth aspects by culturing arecombinant host cell of the eleventh aspect and isolating the OSMRantigen binding protein from said culture.

In a fourteenth aspect, the invention provides OSMR antigen bindingproteins of any one of the first, second, third, fourth, fifth, sixth,sixth, seventh, or eighth aspects that cross-compete with an antibodyselected from the group consisting of:

a) an antibody comprising a light chain comprising the amino acidsequence set forth in SEQ ID:24 and a heavy chain comprising the aminoacid sequence set forth in SEQ ID NO:6;

b) an antibody comprising a light chain comprising the amino acidsequence set forth in SEQ ID:25 and a heavy chain comprising the aminoacid sequence set forth in SEQ ID NO:7; and

c) an antibody comprising a light chain comprising the amino acidsequence set forth in SEQ ID:26 and a heavy chain comprising the aminoacid sequence set forth in SEQ ID NO:8.

DETAILED DESCRIPTION

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All references cited within the body of this specification are expresslyincorporated by reference in their entirety.

Standard techniques may be used for recombinant DNA, oligonucleotidesynthesis, tissue culture and transformation, protein purification, etc.Enzymatic reactions and purification techniques may be performedaccording to the manufacturer's specifications or as commonlyaccomplished in the art or as described herein. The following proceduresand techniques may be generally performed according to conventionalmethods well known in the art and as described in various general andmore specific references that are cited and discussed throughout thespecification. See, e.g., Sambrook et al., 2001, Molecular Cloning: ALaboratory Manuel, 3^(rd) ed., Cold Spring Harbor Laboratory Press, coldSpring Harbor, N.Y., which is incorporated herein by reference for anypurpose. Unless specific definitions are provided, the nomenclature usedin connection with, and the laboratory procedures and techniques of,analytic chemistry, organic chemistry, and medicinal and pharmaceuticalchemistry described herein are those well known and commonly used in theart. Standard techniques may be used for chemical synthesis, chemicalanalyses, pharmaceutical preparation, formulation, and delivery andtreatment of patients.

OSMR

The antigen binding proteins described herein bind to OSMR. OSM andIL-31 signal through OSMR. OSMR is a member of the type I cytokinereceptor family OSMR heterodimerizes with glycoprotein 130 (also knownas gp130, interleukin 6 signal transducer (IL6ST), IL6-beta, or CD130)to form the type II OSMR. OSMR also heterodimerizes with IL-31 receptorA (IL31RA) to form the IL-31 receptor and, thus, transduces OSM- andIL-31-induced signaling events. In exemplary embodiments, an OSMRantigen binding protein binds OSMR and prevents OSM- and/orIL-31-mediated signaling in cells expressing OSMR.

Human OSMR sequences are known in the art. In various aspects, humanOSMR protein sequences are provided in GenBank Accession Nos. AAI25210,AAI25211, NP_003990, and EAW55976. An exemplary human OSMR amino acidsequence (SEQ ID NO: 1) is provided in Table 1. The protein is made upof several domains: Amino acids 1-27 correspond to the signal sequencewhich may be cleaved during processing of the protein in mammaliancells; amino acids 28-740 correspond to the extracellular domain; andamino acids 741-761 correspond to the transmembrane domain. In preferredembodiments, the antigen binding proteins described herein bind to theextracellular domain of OSMR and prevent the interaction of OSM and/orIL-31 with OSMR.

Human OSM sequences are known in the art. In various aspects, human OSMprotein sequences are provided in GenBank Accession Nos. CAG30420,CAG46504, NP_065391, P13725, AAC05173, EAW59864, and AAH11589. Anexemplary human OSM amino acid sequence (SEQ ID NO: 39) is provided inTable 1 Amino acids 1-25 correspond to the signal sequence; amino acids26-220 correspond to the mature protein; and amino acids 221-252correspond to the propeptide sequence.

Human IL-31 sequences are known in the art. In various aspects, humanIL-31 protein sequences are provided in GenBank Accession Nos.NP_001014358, AAS86448, AAI32999, AAI33001, Q6EBC2, and EAW98310. Anexemplary human IL-31 amino acid sequence (SEQ ID NO: 41) is provided inTable 1 Amino acids 1-23 correspond to the putative signal sequence.

Human IL31RA sequences are known in the art. In various aspects, humanIL31RA protein sequences are provided in GenBank Accession Nos.AAS86447, NP_001229567, and CBL94051. An exemplary human IL31RA (v4,isoform 3) amino acid sequence (SEQ ID NO: 43) is provided in Table 1.Amino acids 1-32 correspond to the signal sequence; and amino acids533-553 correspond to the transmembrane sequence.

Human gp130 sequences are known in the art. In various aspects, humangp130 protein sequences are provided in GenBank Accession Nos. AAI17403,AAI17405, EAW54936, NP_002175, ABK41905, and AAA59155. An exemplaryhuman gp130 amino acid sequence (SEQ ID NO: 45) is provided in Table 1.The protein is made up of several domains: Amino acids 1-22 correspondto the signal sequence; amino acids 23-619 correspond to theextracellular domain; amino acids 620-641 correspond to thetransmembrane domain; and amino acids 642-918 correspond to thecytoplasmic domain.

TABLE 1  Human OSMR amino acid sequence (SEQ ID NO: 1)MALFAVFQTTFFLTLLSLRTYQSEVLAERLPLTPVSLKVSTNSTRQSLHLQWTVHNLPYHQELKMVFQIQISRIETSNVIWVGNYSTTVKWNQVLHWSWESELPLECATHFVRIKSLVDDAKFPEPNFWSNWSSWEEVSVQDSTGQDILFVFPKDKLVEEGTNVTICYVSRNIQNNVSCYLEGKQIHGEQLDPHVTAFNLNSVPFIRNKGTNIYCEASQGNVSEGMKGIVLFVSKVLEEPKDFSCETEDFKTLHCTWDPGTDTALGWSKQPSQSYTLFESFSGEKKLCTHKNWCNWQITQDSQETYNFTLIAENYLRKRSVNILFNLTHRVYLMNPFSVNFENVNATNAIMTWKVHSIRNNFTYLCQIELHGEGKMMQYNVSIKVNGEYFLSELEPATEYMARVRCADASHFWKWSEWSGQNFTTLEAAPSEAPDVWRIVSLEPGNHTVTLFWKPLSKLHANGKILFYNVVVENLDKPSSSELHSIPAPANSTKLILDRCSYQICVIANNSVGASPASVIVISADPENKEVEEERIAGTEGGFSLSWKPQPGDVIGYVVDWCDHTQDVLGDFQWKNVGPNTTSTVISTDAFRPGVRYDFRIYGLSTKRIACLLEKKTGYSQELAPSDNPHVLVDTLTSHSFTLSWKDYSTESQPGFIQGYHVYLKSKARQCHPRFEKAVLSDGSECCKYKIDNPEEKALIVDNLKPESFYEFFITPFTSAGEGPSATFTKVTTPDEHSSMLIHILLPMVFCVLLIMVMCYLKSQWIKETCYPDIPDPYKSSILSLIKFKENPHLIIMNVSDCIPDAIEVVSKPEGTKIQFLGTRKSLTETELTKPNYLYLLPTEKNHSGPGPCICFENLTYNQAASDSGSCGHVPVSPKAPSMLGLMTSPENVLKALEKNYMNSLGEIPAGETSLNYVSQLASPMFGDKDSLPTNPVEAPHCSEYKMQMAVSLRLALPPPTENSSLSSITLLDPGEHYCHuman OSM amino acid sequence (SEQ ID NO: 39)MGVLLTQRTLLSLVLALLFPSMASMAAIGSCSKEYRVLLGQLQKQTDLMQDTSRLLDPYIRIQGLDVPKLREHCRERPGAFPSEETLRGLGRRGFLQTLNATLGCVLHRLADLEQRLPKAQDLERSGLNIEDLEKLQMARPNILGLRNNIYCMAQLLDNSDTAEPTKAGRGASQPPTPTPASDAFQRKLEGCRFLHGYHRFMHSVGRVFSKWGESPNRSRRHSPHQALRKGVRRTRPSRKGKRLMTRGQLPR Human IL-31 amino acid sequence (SEQ ID NO: 41)MASHSGPSTSVLFLFCCLGGWLASHTLPVRLLRPSDDVQKIVEELQSLSKMLLKDVEEEKGVLVSQNYTLPCLSPDAQPPNNIHSPAIRAYLKTIRQLDNKSVIDEIIEHLDKLIFQDAPETNISVPTDTHECKRFILTISQQFSECMDLALKSLTSGAQQATTHuman IL31RA amino acid sequence (SEQ ID NO: 43)MKLSPQPSCVNLGMMWTWALWMLPSLCKFSLAALPAKPENISCVYYYRKNLTCTWSPGKETSYTQYTVKRTYAFGEKHDNCTTNSSTSENRASCSFFLPRITIPDNYTIEVEAENGDGVIKSHMTYWRLENIAKTEPPKIFRVKPVLGIKRMIQIEWIKPELAPVSSDLKYTLRFRTVNSTSWMEVNFAKNRKDKNQTYNLTGLQPFTEYVIALRCAVKESKFWSDWSQEKMGMTEEEAPCGLELWRVLKPAEADGRRPVRLLWKKARGAPVLEKTLGYNIWYYPESNTNLTETMNTTNQQLELHLGGESFWVSMISYNSLGKSPVATLRIPAIQEKSFQCIEVMQACVAEDQLVVKWQSSALDVNTWMIEWFPDVDSEPTTLSWESVSQATNWTIQQDKLKPFWCYNISVYPMLHDKVGEPYSIQAYAKEGVPSEGPETKVENIGVKTVTITWKEIPKSERKGIICNYTIFYQAEGGKGFSKTVNSSILQYGLESLKRKTSYIVQVMASTSAGGTNGTSINFKTLSFSVFEIILITSLIGGGLLILIILTVAYGLKKPNKLTHLCWPTVPNPAESSIATWHGDDFKDKLNLKESDDSVNTEDRILKPCSTPSDKLVIDKLVVNFGNVLQEIFTDEARTGQENNLGGEKNGTRILSSCPTSI Human gp130 amino acid sequence (SEQ ID NO: 45)MLTLQTWLVQALFIFLTTESTGELLDPCGYISPESPVVQLHSNFTAVCVLKEKCMDYFHVNANYIVWKTNHFTIPKEQYTIINRTASSVTFTDIASLNIQLTCNILTFGQLEQNVYGITIISGLPPEKPKNLSCIVNEGKKMRCEWDGGRETHLETNFTLKSEWATHKFADCKAKRDTPTSCTVDYSTVYFVNIEVWVEAENALGKVTSDHINFDPVYKVKPNPPHNLSVINSEELSSILKLTWTNPSIKSVIILKYNIQYRTKDASTWSQIPPEDTASTRSSFTVQDLKPFTEYVFRIRCMKEDGKGYWSDWSEEASGITYEDRPSKAPSFWYKIDPSHTQGYRTVQLVWKTLPPFEANGKILDYEVTLTRWKSHLQNYTVNATKLTVNLTNDRYLATLTVRNLVGKSDAAVLTIPACDFQATHPVMDLKAFPKDNMLWVEWTTPRESVKKYILEWCVLSDKAPCITDWQQEDGTVHRTYLRGNLAESKCYLITVTPVYADGPGSPESIKAYLKQAPPSKGPTVRTKKVGKNEAVLEWDQLPVDVQNGFIRNYTIFYRTIIGNETAVNVDSSHTEYTLSSLTSDTLYMVRMAAYTDEGGKDGPEFTFTTPKFAQGEIEAIVVPVCLAFLLTTLLGVLFCFNKRDLIKKHIWPNVPDPSKSHIAQWSPHTPPRHNFNSKDQMYSDSNFTDVSVVEIVANDKKPFPEDLKSLDLFKKEKINTEGHSSGIGGSSCMSSSRPSISSSDENESSQNTSSTVQYSTVVHSGYRHQVPSVQVFSRSESTQPLLDSEERPEDLQLVDHVDGGDGILPRQQYFKQNCSQHESSPDISHFERSKQVSSVNEEDFVRLKQQISDHISQSCGSGQMKMFQEVSAADAFGPGTEGQVERFETVGMEAATDEGMPKSYLPQTVRQGGYMPQ

In particular embodiments of the present invention, antigen bindingproteins described herein bind both human and cynomolgus monkey OSMRwith high affinity, including those that bind with high affinity andblock interaction of cynomolgus monkey OSM and/or IL-31 to cynomolgusmonkey OSMR. These characteristics allow informative toxicology studiesin non-human primates.

A Rhesus macaque (Macaca mulatta) OSMR protein sequence is known in theart and is provided in GenBank Accession No. XP_001083745. An exemplarycynomolgus monkey (Macaca fascicularis) OSMR amino acid sequence (SEQ IDNO:2) is provided in Table 2. The protein is made up of several domains:Amino acids 1-27 correspond to the signal sequence which may be cleavedduring processing of the protein in mammalian cells; amino acids 28-737correspond to the extracellular domain; and amino acids 738-757correspond to the transmembrane domain. In preferred embodiments, theantigen binding proteins described herein bind to the extracellulardomain of OSMR and prevent the interaction of OSM and/or IL-31 withOSMR.

A Rhesus macaque (Macaca mulatta) OSM protein sequence is known in theart and is provided in GenBank Accession No. NP_001181403. An exemplarycynomolgus monkey (Macaca fascicularis) OSM amino acid sequence (SEQ IDNO:40) is provided in Table 2. Amino acids 1-196 correspond to themature cynomolgus OSM.

A Rhesus macaque (Macaca mulatta) IL-31 protein sequence is known in theart and is provided in GenBank Accession No. XP_001096743. An exemplarycynomolgus monkey (Macaca fascicularis) IL-31 amino acid sequence (SEQID NO:42) is provided in Table 2. This sequence represents the maturecynomolgus monkey IL-31.

An exemplary cynomolgus monkey (Macaca fascicularis) IL31RA amino acidsequence (SEQ ID NO: 44) is provided in Table 2 Amino acids 1-19correspond to the signal sequence; and amino acids 520-540 correspond tothe transmembrane domain.

A Rhesus macaque (Macaca mulatta) gp130 protein sequence is known in theart and is provided in GenBank Accession No. NP_001252920. An exemplarycynomolgus monkey (Macaca fascicularis) gp130 amino acid sequence (SEQID NO: 46) is provided in Table 2. The protein is made up of severaldomains: Amino acids 1-22 correspond to the signal sequence; amino acids23-619 correspond to the extracellular domain; amino acids 620-641correspond to the transmembrane domain; and amino acids 642-918correspond to the cytoplasmic domain.

TABLE 2  Cynomolgus monkey OSMR amino acid sequence (SEQ ID NO: 2)MALFVVFQTTFFLILLSLRTYQSEVLAERLPLTPVSLKVSTNSIHQSLHLQWTVHNLPYHQELKMVFQIQISRIETSNVVWVGNYSTPVKWNQVLHWSWESELPLECATHFVRIKSVIDDASFPEPNFWSNWSSWEEVSVQDYLGRGTLFVFPKDKLVEEGSNVTICYVSRNIQNNVSCYLEGKQIHGEQLDPHVTAFNLNSVPFIRNRGTNIYCEASQGNVSKGIEGIVLFVSKVLEEPKDFSCESQDFKTLHCTWDPGTDTALGWSKQPSQSYTLFESFSGEKKLCTHKNWCNWQITQDSQEMYNFTLIAENYLRKRSVNILFNLTHRVYLMNPFSVNFENVNATNAIMTWKVHSMRNNFTYLCQIELHGEGKMMQYDVSINVNGEYFLSELEPATEYMARVRCADASHFWKWTEWSGQNFTTLEAAPSEAPDVWRSVNSEPGNHTVTLFWKPLSKLHANGKILFYNVVVENLDKPSRSELRSIPAPANSTKLILDRCSYQICVTANNSVGASPASIIVISADPENKEVEEERIAGTEGGFSLSWKPQPGDVIGYVVDWCDHPQDVLQWKNVGPNTTSTVISTDAFRPGVRYDPRIYGLSTKRIACLLEKKTGYSQELAPSDNPHYLVDMLTSHSFTLSWKDYSTESQPGFIQGYHVYLKSKARQCHPRFQKAVLSDGSECCRYKIDNPEEKALIVDNLKPESFYEFFVTPFTSAGEGPNATFTKVTTPDEHSSMLIRILLPMVFCVLLIMIVCYLKSQWIKETCYPDIPDPYKSSILSLIKFKENPHLTIMNVSDCIPDAIEVVSKPEGTKIQLLGTRKSLTETELTKPNYLYLLPTEKNHSGPGPCICFENFTYNQAASDAGSCGHVPVPPKAPPSMLGLMTSPENVLKALEKNYMNSLGEVPAGETSLNYVSQLASPMSGDKDSLPTNPVEPPHCSEYKMQMAVPLRLALPPPTENSSLSSITLLDPGEHYRCynomolgus monkey OSM amino acid sequence (SEQ ID NO: 40)AAMGSCSKEYRMLLGQLQKQTDLMQDTSRLLDPYIRIQGLDIPKLREHCRESPGAFPSEETLRGLGRRGFLQTLNATLGRILHRLADLEQHLPKAQDLERSGLNIEDLEKLQMARPNVLGLRNNVYCMAQLLDNSDMTEPTKAGRGTPQPPTPTPTSDVFQRKLEGCSFLRGYHRFMHSVGRVFSKWGESPNRSRRCynomolgus monkey IL-31 amino acid sequence (SEQ ID NO: 42)TLPVHFLQPSDIQKIVEELQSLSKMLLKD VKEDKGVLVSQNYTLPCLTPDAQPPNIIHSPAIRAYLKTIRQLDNKSVIDEIIEHLDKLIFQDAPETNISVPTDTHECKRFILTISQQFSECMDLALKSLTSGAQQATTCynomolgus monkey IL31RA amino acid sequence (SEQ ID NO: 44)MMWTWALWMFPLLCKFGLAALPAKPENISCVYYYRKNLTCTWSPGKETSYTQYTAKRTYAFGKKHDNCTTSSSTSENRASCSFFLPRITIPDNYTIEVEAENGDGVIKSDMTCWRLEDIAKTEPPEIFSVKPVLGIKRMIRIEWIKPELAPVSSDLKYALRFRTVNSTSWMEVNFAKNRKDTNQTYNLMGLQAFTEYVVALRCAVKESKFWSDWSQEKMGMTEEEAPCGLELWRVLKPTEVDGRRPVRLLWKKARGAPVLEKTLGYNIWYFPENNTNLTETVNTTNQQLELHLGGESYWVSMISYNSLGKSPVTTLRIPAIQEKSFRCIEVMQACLAEDQLVVKWQSSALDVNTWMIEWFPDMDSEHPTLSWESVSQATNWTIQQDKLKPFWCYNISVYPMLHDKVGEPYSIQAYAKEGIPSKGPETKVENIGVKTVTITWKEIPKSERKGIICNYTIFYQAEGGTGFSKTVNSSILQYGLESLKRKTSYTVRVMASTSAGGINGTSINFKTLSFSVPHILITSLIGGGLLILIILTVAYGLKKPNKLTHLCWPSVPNPAESSIATWRGDDFKD KLNLKESDDSVNTEDRILKPCSTPSDKLVIDKSVVNFGNVLQEMFTDEARTGQENNLGGEKNENRILSSC PTSICynomolgus monkey gp130 amino acid sequence (SEQ ID NO: 46)MLTLQTWVVQALFIFLTTESIGELLDPCGYISPESPVVQLHSNFTAVCVLKEKCMDYFHVNANYIVWKTNHFTIPKEQYTIINRTASSVTFTDISSLNIQLTCNILTFGQLEQNVYGITIISGLPPEKPKNLSCIVNEGKKMRCEWNRGRETHLETNFTLKSEWATHKFADCKAKRDTPTSCTVDYSTVYFVNIEVWVEAENALGKVTSDHINFDPVYKVKPNPPHNLSVINSEELSSILKLTWTNPSIKSVIRLKYNIQYRTKDASTWSQIPPEDTASTRSSFTVQDLKPFTEYVFRICCMKEDGKGYWSDWSEEANGITYEDRPSKAPSFWYKIDPSHAQGYRTVQLMWKTLPPFEANGKILDYEVTLTRWKSHLQNYTVNDTKLTVNLTNDRYVATLTARNLVGKSDAAVLTIPACDFQATHPVMDLKAFPKDNMLWVEWTTPRESVKKYILEWCVLSDKAPCIADWQQEDGTVHRTHLRGNLAESKCYLITVTPVYADGPGSPESIKAYLKQAPPSKGPTVRTKKVGKNEAVLEWDQLPVDVQNGFIRNYTIFYRTIIGNETAVNVDSSHTEYTLSSLTSDTLYMVRMAAYTDEGGKDGPEFTFTTPKFAQGEIEAIVVPVCLAFLLTTLLGVLFCFNKRDLIKKHIWPNVPDPSKSHIAQWSPHTPPRHNFSSKDQMYSDGNFTDVSVVEIEANDKKPFPEDLKSLDLFKKEKINTEGHSSGIGGSSCMSSSRPSISSSDENESSQNTSSTVQYSTVVHSGYRHQVPSVQVFSRSESTQPLLDSEERPEDLQLVDHVDGSDDILPRQQYFKQNCSQHESSPDISHFERSKQVSSVNEEDFVRLKQQISDHISQSCGSGEMKMFQEVSAADPFGPGTEGQVERFETIGMEAAIDEGMPKSYLPQTVRQGGYMPQ

OSMR Antigen Binding Proteins

The present invention provides antigen binding proteins thatspecifically bind OSMR. Embodiments of antigen binding proteins comprisepeptides and/or polypeptides that specifically bind OSMR. Such peptidesor polypeptides may optionally include one or more port-translationalmodifications. Embodiments of antigen binding proteins includeantibodies and fragments thereof, as variously defined herein, thatspecifically bind OSMR. These include antibodies that specifically bindhuman OSMR, including those that inhibit OSM and/or IL-31 from bindingand/or activating OSMR.

The antigen binding proteins of the invention specifically bind to OSMR.“Specifically binds” as used herein means that the antigen bindingprotein preferentially binds OSMR over other proteins. In someembodiments “specifically binds” means the OSMR antigen binding proteinhas a higher affinity for OSMR than for other proteins. OSMR antigenbinding proteins that specifically bind OSMR may have a binding affinityfor human OSMR of less than or equal to 1×10⁻⁷ M, less than or equal to2×10⁻⁷ M, less than or equal to 3×10⁻⁷ M, less than or equal to 4×10⁻⁷M, less than or equal to 5×10⁻⁷ M, less than or equal to 6×10⁻⁷ M, lessthan or equal to 7×10⁻⁷ M, less than or equal to 8×10⁻⁷ M, less than orequal to 9×10⁻⁷ M, less than or equal to 1×10⁻⁸ M, less than or equal to2×10⁻⁸ M, less than or equal to 3×10⁻⁸ M, less than or equal to 4×10⁻⁸M, less than or equal to 5×10⁻⁸ M, less than or equal to 6×10⁻⁸ M, lessthan or equal to 7×10⁻⁸ M, less than or equal to 8×10⁻⁸ M, less than orequal to 9×10⁻⁸ M, less than or equal to 1×10⁻⁹ M, less than or equal to2×10⁻⁹ M, less than or equal to 3×10⁻⁹ M, less than or equal to 4×10⁻⁹M, less than or equal to 5×10⁻⁹ M, less than or equal to 6×10⁻⁹ M, lessthan or equal to 7×10⁻⁹ M, less than or equal to 8×10⁻⁹ M, less than orequal to 9×10⁻⁹ M, less than or equal to 1×10⁻¹⁰ M, less than or equalto 2×10⁻¹⁰ M, less than or equal to 3×10⁻¹⁰ M, less than or equal to4×10⁻¹⁰ M, less than or equal to 5×10⁻¹⁰ M, less than or equal to6×10⁻¹⁰ M, less than or equal to 7×10⁻¹⁰ M, less than or equal to8×10⁻¹⁰ M, less than or equal to 9×10⁻¹⁰ M, less than or equal to1×10⁻¹¹ M, less than or equal to 2×10⁻¹¹ M, less than or equal to3×10⁻¹¹ M, less than or equal to 4×10⁻¹¹ M, less than or equal to5×10⁻¹¹ M, less than or equal to 6×10⁻¹¹ M, less than or equal to7×10⁻¹¹ M, less than or equal to 8×10⁻¹¹ M, less than or equal to9×10⁻¹¹ M, less than or equal to 1×10⁻¹² M, less than or equal to2×10⁻¹² M, less than or equal to 3×10⁻¹² M, less than or equal to4×10⁻¹² M, less than or equal to 5×10⁻¹² M, less than or equal to6×10⁻¹² M, less than or equal to 7×10⁻¹² M, less than or equal to8×10⁻¹² M, or less than or equal to 9×10⁻¹² M.

Methods of measuring the binding affinity of an antigen binding proteinare well known in the art. Methods in common use for affinitydetermination include Surface Plasmon Resonance (SPR) (Morton and Myszka“Kinetic analysis of macromolecular interactions using surface plasmonresonance biosensors” Methods in Enzymology (1998) 295, 268-294),Bio-Layer Interferometry, (Abdiche et al “Determining Kinetics andAffinities of Protein Interactions Using a Parallel Real-time Label-freeBiosensor, the Octet” Analytical Biochemistry (2008) 377, 209-217),Kinetic Exclusion Assay (KinExA) (Darling and Brault “Kinetic exclusionassay technology: characterization of molecular interactions” Assay andDrug Dev Tech (2004) 2, 647-657), isothermal calorimetry (Pierce et al“Isothermal Titration calorimetry of Protein-Protein Interactions”Methods (1999) 19, 213-221) and analytical ultracentrifugation (Lebowitzet al “Modern analytical ultracentrifugation in protein science: Atutorial review” Protein Science (2002), 11:2067-2079). Example 5provides exemplary methods of affinity determination.

It is understood that when reference is made to the various embodimentsof the OSMR-binding antibodies herein, that it also encompassesOSMR-binding fragments thereof. An OSMR-binding fragment comprises anyof the antibody fragments or domains described herein that retains theability to specifically bind to OSMR. The OSMR-binding fragment may bein any of the scaffolds described herein.

In certain therapeutic embodiments, an OSMR antigen binding proteininhibits binding of OSM and/or IL-31 to OSMR and/or inhibits one or morebiological activities associated with the binding of OSM and/or IL-31 toOSMR, e.g., OSM- and/or IL-31-mediated signaling. Such antigen bindingproteins are said to be “neutralizing.” In certain embodiments, theneutralizing OSMR antigen binding protein specifically binds OSMR andinhibits binding of OSM and/or IL-31 to OSMR from anywhere between 10%to 100%, such as by at least about 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% ormore. For example, OSMR antigen binding proteins may be tested forneutralizing ability by determining the ability of the antigen bindingprotein to block binding of OSM and/or IL-31 to OSMR, see, e.g., thehuman OSMR and cynomolgus OSMR blocking assays of Examples 2 and 3,respectively. Alternatively, OSMR antigen binding proteins may be testedfor neutralizing ability in an assay that measures the effect of thepresence of the OSMR antigen binding protein in an assay measuring OSM-and/or IL-31-mediated biological function. For example, the ability ofOSM to induce a biological response, such as stimulation of plasminogenactivator activity in cultured bovine aortic endothelial cells,regulation of IL-6 expression in human endothelial cells, andstimulation of LDL uptake and up-regulation of cell surface LDLreceptors in HepG2 cells. Alternatively, the ability of IL-31 to induceinflammation in the skin.

Embodiments of antigen binding proteins comprise a scaffold structure,as variously defined herein, with one or more complementaritydetermining regions (CDRs). Embodiments further include antigen bindingproteins comprising a scaffold structure with one or more antibodyvariable domains, either heavy or light. Embodiments include antibodiesthat comprise a light chain variable domain selected from the groupconsisting of Ab1 Light Chain Variable Domain (LCv), Ab2 LCv, and Ab3LCv (SEQ ID NOS:27-29, respectively) and/or a heavy chain variabledomain selected from the group consisting of Ab1 Heavy Chain VariableDomain (HCv), Ab2 HCv, and Ab3 HCv (SEQ ID NOS:9-11, respectively), andfragments, derivatives, muteins, and variants thereof.

An exemplary heavy chain variable domain variant of SEQ ID NO:9 containsan amino acid other than asparagine (for example, aspartic acid) at theposition corresponding to position 73 in SEQ ID NO:9. The amino acidsequence set forth in SEQ ID NO:53 is an example of a heavy chainvariable domain variant of SEQ ID NO:9.An exemplary heavy chain variable domain variant of SEQ ID NO:10contains an amino acid other than asparagine (for example, asparticacid) at the position corresponding to position 73 in SEQ ID NO:10. Theamino acid sequence set forth in SEQ ID NO:54 is an example of a heavychain variable domain variant of SEQ ID NO:10.

An exemplary light chain comprising Ab1 LCv is a light chain comprisingthe amino acid sequence set forth in SEQ ID NO:24.

An exemplary light chain comprising Ab2 LCv is a light chain comprisingthe amino acid sequence set forth in SEQ ID NO:25.

An exemplary light chain comprising Ab3 LCv is a light chain comprisingthe amino acid sequence set forth in SEQ ID NO:26.

An exemplary heavy chain comprising Ab1 HCv is a heavy chain comprisingthe amino acid sequence set forth in SEQ ID NO:6.

An exemplary heavy chain comprising a variant of Ab1 HCv is a heavychain comprising the amino acid sequence set forth in SEQ ID NO:50.

An exemplary heavy chain comprising Ab2 HCv is a heavy chain comprisingthe amino acid sequence set forth in SEQ ID NO:7.

An exemplary heavy chain comprising a variant of Ab2 HCv is a heavychain comprising the amino acid sequence set forth in SEQ ID NO:51.

An exemplary heavy chain comprising Ab3 HCv is a heavy chain comprisingthe amino acid sequence set forth in SEQ ID NO:8.

An exemplary heavy chain comprising a variant of Ab3 HCv is a heavychain comprising the amino acid sequence set forth in SEQ ID NO:52.

Additional examples of scaffolds that are envisioned include, but arenot limited to: fibronectin, neocarzinostatin CBM4-2, lipocalins, T-cellreceptor, protein-A domain (protein Z), Im9, TPR proteins, zinc fingerdomains, pVIII, avian pancreatic polypeptide, GCN4, WW domain Srchomology domain 3, PDZ domains, TEM-1 beta-lactamase, thioredoxin,staphylococcal nuclease, PHD-finger domains, CL-2, BPTI, APPI, HPSTI,ecotin, LACI-D1, LDTI, MTI-II, scorpion toxins, insect defensin-Apeptide, EETI-II, Min-23, CBD, PBP, cytochrome b-562, Ldl receptordomains, gamma-crystallin, ubiquitin, transferrin, and or C-typelectin-like domains. Non-antibody scaffolds and their use astherapeutics are reviewed in Gebauer and Skerra, Curr. Opin. Chem.Biol., 13:245-255 (2009) and Binz et al., Nat. Biotech., 23(10):1257-68(2005), which are incorporated herein by reference in its entirety.

Aspects of the invention include antibodies comprising the followingvariable domains: Ab1 LCv/Ab1 HCv (SEQ ID NO:27/SEQ ID NO:9), Ab2LCv/Ab2 HCv (SEQ ID NO:28/SEQ ID NO:10), Ab3 LCv/Ab3 HCv (SEQ IDNO:29/SEQ ID NO:11), and combinations thereof, as well as fragments,derivatives, muteins and variants thereof.

Also included are antibodies comprising the following variable domains:SEQ ID NO:27/SEQ ID NO:53; and SEQ ID NO:28/SEQ ID NO:54.

Exemplary antibodies of the invention include Ab1 (SEQ ID NO:24/SEQ IDNO:6), Ab2 (SEQ ID NO:25/SEQ ID NO:7), and Ab3 (SEQ ID NO:26/SEQ IDNO:8).

Additional exemplary antibodies include: SEQ ID NO:24/SEQ ID NO:50; SEQID NO:25/SEQ ID NO:51; and SEQ ID NO:26/SEQ ID NO:52.

Typically, each variable domain of an antibody light or heavy chaincomprises three CDRs. The heavy chain variable domain comprises a heavychain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3(HCDR3). The light chain variable domain comprises a light chain CDR1(LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3). Incertain embodiments, an antigen binding protein comprises one or moreCDRs contained within the preferred variable domains described herein.

Examples of such CDRs include, but are not limited to:

the CDRs of Ab1 LCv: LCDR1 (SEQ ID NO:30), LCDR2 (SEQ ID NO:33), andLCDR3 (SEQ ID NO:36);

the CDRs of Ab2 LCv: LCDR1 (SEQ ID NO:31), LCDR2 (SEQ ID NO:34), andLCDR3 (SEQ ID NO:37);

the CDRs of Ab3 LCv: LCDR1 (SEQ ID NO:32), LCDR2 (SEQ ID NO:35), andLCDR3 (SEQ ID NO:38);

the CDRs of Ab1 HCv: HCDR1 (SEQ ID NO:12), HCDR2 (SEQ ID NO:15), andHCDR3 (SEQ ID NO:18);

the CDRs of Ab2 HCv: HCDR1 (SEQ ID NO:13), HCDR2 (SEQ ID NO:16), andHCDR3 (SEQ ID NO:19); and

the CDRs of Ab3 HCv: HCDR1 (SEQ ID NO:14), HCDR2 (SEQ ID NO:17), andHCDR3 (SEQ ID NO:20).

In some embodiments, the antigen binding protein comprises: A) apolypeptide, e.g., a light chain, that comprises an LCDR1 having anamino acid sequence selected from the group consisting of SEQ ID NOS:30,31, and 32; an LCDR2 having an amino acid sequence selected from thegroup consisting of SEQ ID NOS:33, 34, and 35; and/or an LCDR3 having anamino acid sequence selected from the group consisting of SEQ ID NOS:36,37, and 38; and/or B) a polypeptide, e.g., a heavy chain, that comprisesan HCDR1 having an amino acid sequence selected from the groupconsisting of SEQ ID NOS:12, 13, and 14; an HCDR2 having an amino acidsequence selected from the group consisting of SEQ ID NOS:15, 16, and17; and/or an HCDR3 having an amino acid sequence selected from thegroup consisting of SEQ ID NOS:18, 19, and 20.

In further embodiments, the antigen binding protein comprise A) a lightchain amino acid sequence that comprises a LCDR1, LCDR2, and LCDR3 ofany of Ab1 LCv, Ab2 LCv, and Ab3 LCv and B) a heavy chain amino acidsequence that comprises a HCDR1, HCDR2, and HCDR3 of any of Ab1 HCv, Ab2HCv, and Ab3 HCv.

In certain embodiments, the CDRs include no more than one, no more thantwo, no more than three, no more than four, no more than five, or nomore than six amino acid additions, deletions, or substitutions from anexemplary CDR set forth herein.

Aspects of the invention include antibodies comprising a light chainvariable domain selected from the group consisting of SEQ ID NOS:27, 28,and 29. Aspects of the invention include antibodies comprising a heavychain variable domain selected from the group consisting of SEQ IDNOS:9, 10, and 11. Further aspects of the invention include antibodiescomprising A) a light chain variable domain selected from the groupconsisting of SEQ ID NOS:27, 28, and 29, and B) a heavy chain variabledomain selected from the group consisting of SEQ ID NOS:9, 10, and 11.

Antibodies of the invention can comprise any constant region known inthe art. The light chain constant region can be, for example, a kappa-or lambda-type light chain constant region, e.g., a human kappa- orlambda-type light chain constant region. The heavy chain constant regioncan be, for example, an alpha-, delta-, epsilon-, gamma-, or mu-typeheavy chain constant region, e.g., a human alpha-, delta-, epsilon-,gamma-, or mu-type heavy chain constant region. In one embodiment thelight or heavy chain constant region is a fragment, derivative, variant,or mutein of a naturally occurring constant region.

Aspects of the invention include antibodies comprising a light chainvariable region selected from the group consisting of SEQ ID NOS:27, 28,and 29 having no more than one, no more than two, no more than three, nomore than four, no more than five, no more than six, no more than seven,no more than eight, no more than nine, or no more than ten amino acidadditions, deletions, or substitutions. Aspects of the invention includeantibodies comprising a heavy chain variable region selected from thegroup consisting of SEQ ID NOS:9, 10, and 11 having no more than one, nomore than two, no more than three, no more than four, no more than five,no more than six, no more than seven, no more than eight, no more thannine, or no more than ten amino acid additions, deletions, orsubstitutions. Further aspects of the invention include antibodiescomprising A) comprising a light chain variable region selected from thegroup consisting of SEQ ID NOS:27, 28, and 29 having no more than one,no more than two, no more than three, no more than four, no more thanfive, no more than six, no more than seven, no more than eight, no morethan nine, or no more than ten amino acid additions, deletions, orsubstitutions, and B) a heavy chain variable region selected from thegroup consisting of SEQ ID NOS:9, 10, and 11 having no more than one, nomore than two, no more than three, no more than four, no more than five,no more than six, no more than seven, no more than eight, no more thannine, or no more than ten amino acid additions, deletions, orsubstitutions.

In one variation, the antigen binding protein comprises an amino acidsequence that is at least 80%, at least 81%, at least 82%, at least 83%,at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% identical to a light chain variable region amino acid sequenceselected from the group consisting of SEQ ID NOS:27, 28, and 29. Inanother variation, the antigen binding protein comprises an amino acidsequence that is at least 80%, at least 81%, at least 82%, at least 83%,at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% identical to a heavy chain variable region amino acid sequenceselected from the group consisting of SEQ ID NOS:9, 10, and 11. In yet afurther embodiment, the antigen binding protein comprises A) an aminoacid sequence that is at least 80%, at least 81%, at least 82%, at least83%, at least 84%, at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% identical to a light chain variable region aminoacid sequence selected from the group consisting of SEQ ID NOS:27, 28,and 29, and B) an amino acid sequence that is at least 80%, at least81%, at least 82%, at least 83%, at least 84%, at least 85%, at least86%, at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% identical to a heavychain variable region amino acid sequence selected from the groupconsisting of SEQ ID NOS:9, 10, and 11.

OSMR antigen binding proteins comprising a heavy chain variable domainhaving the above-defined sequence relatedness to SEQ ID NO:9 canoptionally contain an amino acid other than asparagine (for example,aspartic acid) at the position corresponding to position 73 in SEQ IDNO:9. In such embodiments, the heavy chain variable domain optionallycomprises the amino acid sequence set forth in SEQ ID NO:53.OSMR antigen binding proteins comprising a heavy chain variable domainhaving the above-defined sequence relatedness to SEQ ID NO:10 canoptionally contain an amino acid other than asparagine (for example,aspartic acid) at the position corresponding to position 73 in SEQ IDNO:10. In such embodiments, the heavy chain variable domain optionallycomprises the amino acid sequence set forth in SEQ ID NO:54.

In certain embodiments, the antigen binding protein comprises a lightchain and/or heavy chain CDR3. In some embodiments, the antigen bindingprotein comprises an amino acid sequence selected from the group ofsequences set forth in SEQ ID NOS:36, 37, 38, 18, 19, and 20. In certainembodiments, the amino acid sequence includes no more than one, no morethan two, no more than three, no more than four, no more than five, orno more than six amino acid additions, deletions, or substitutions fromthe exemplary sequence set forth in SEQ ID NOS:36, 37, 38, 18, 19, and20. Thus, embodiments of the invention include antigen binding proteincomprising an amino acid sequence that is at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% identical to an amino acidsequence selected from the group of sequences set forth in SEQ IDNOS:36, 37, 38, 18, 19, and 20.

In certain embodiments, the antigen binding protein comprises a lightchain and/or heavy chain CDR2. In some embodiments, the antigen bindingprotein comprises an amino acid sequence selected from the group ofsequences set forth in SEQ ID NOS:33, 34, 35, 15, 16, and 17. In certainembodiments, the amino acid sequence includes no more than one, no morethan two, no more than three, no more than four, no more than five, orno more than six amino acid additions, deletions, or substitutions fromthe exemplary sequence set forth in SEQ ID NOS:33, 34, 35, 15, 16, and17. Thus, embodiments of the invention include antigen binding proteincomprising an amino acid sequence that is at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% identical to an amino acidsequence selected from the group of sequences set forth in SEQ IDNOS:33, 34, 35, 15, 16, and 17.

In certain embodiments, the antigen binding protein comprises a lightchain and/or heavy chain CDR1. In some embodiments, the antigen bindingprotein comprises an amino acid sequence selected from the group ofsequences set forth in SEQ ID NOS:30, 31, 32, 12, 13, and 14. In certainembodiments, the amino acid sequence includes no more than one, no morethan two, no more than three, no more than four, no more than five, orno more than six amino acid additions, deletions, or substitutions fromthe exemplary sequence set forth in SEQ ID NOS:30, 31, 32, 12, 13, and14. Thus, embodiments of the invention include antigen binding proteincomprising an amino acid sequence that is at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% identical to an amino acidsequence selected from the group of sequences set forth in SEQ IDNOS:30, 31, 32, 12, 13, and 14.

The antigen binding proteins of the invention comprise the scaffolds oftraditional antibodies, including human and monoclonal antibodies,bispecific antibodies, diabodies, minibodies, domain antibodies,synthetic antibodies (sometimes referred to herein as “antibodymimetics”), chimeric antibodies, antibody fusions (sometimes referred toas “antibody conjugates”), and fragments of each, respectively. Theabove described CDRs, including various combinations of the CDRs, may begrafted into any of the following scaffolds.

As used herein, the term “antibody” refers to the various forms ofmonomeric or multimeric proteins comprising one or more polypeptidechains that specifically binds to an antigen, as variously describedherein. In certain embodiments, antibodies are produced by recombinantDNA techniques. In additional embodiments, antibodies are produced byenzymatic or chemical cleavage of naturally occurring antibodies. Inanother aspect, the antibody is selected from the group consisting of:a) a human antibody; b) a humanized antibody; c) a chimeric antibody; d)a monoclonal antibody; e) a polyclonal antibody; f) a recombinantantibody; g) an antigen-binding fragment; h) a single chain antibody; i)a diabody; j) a triabody, k) a tetrabody, l) a Fab fragment; m) aF(ab′)₂ fragment, n) an IgA antibody, o) an IgD antibody, p) an IgEantibody, q) an IgG1 antibody, r) an IgG2 antibody, s) an IgG3 antibody,t) an IgG4 antibody, and u) an IgM antibody.

A variable region or domain comprises at least three heavy or lightchain CDRs embedded within a framework region (designated frameworkregions FR1, FR2, FR3, and FR4). Kabat et al., 1991, Sequences ofProteins of Immunological Interest, Public Health Service N.I.H.,Bethesda, Md. Traditional antibody structural units typically comprise atetramer. Each tetramer is typically composed of two identical pairs ofpolypeptide chains, each pair having one “light” and one “heavy” chain.The amino-terminal portion of each chain includes a variable region ofabout 100 to 110 or more amino acids primarily responsible for antigenrecognition. The carboxy-terminal portion of each chain defines aconstant region primarily responsible for effector function. Human lightchains are classified as kappa or lambda light chains. Heavy chains areclassified as mu, delta, gamma, alpha, or epsilon, and define theantibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG hasseveral subclasses, including, but not limited to IgG1, IgG2, IgG3, andIgG4. IgM has subclasses, including, but not limited to IgM1 and IgM2.Embodiments of the invention include all such classes and subclasses ofantibodies that incorporate a variable domain or CDR of the antigenbinding proteins, as described herein.

Some naturally occurring antibodies, such as those found in camels andllamas, are dimers consisting of two heavy chains and include no lightchains. The invention encompasses dimeric antibodies of two heavychains, or fragments thereof, that can bind to OSMR.

The variable regions of the heavy and light chains typically exhibit thesame general structure of relatively conserved framework regions (FR)joined by three hypervariable regions, i.e., the complementaritydetermining regions or CDRs. The CDRs are primarily responsible forantigen recognition and binding. The CDRs from the two chains of eachpair are aligned by the framework regions, enabling binding to aspecific epitope. From N-terminal to C-terminal, both light and heavychains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.The assignment of amino acids to each domain is in accordance with thedefinitions of Kabat.

CDRs constitute the major surface contact points for antigen binding.The CDR3 or the light chain and, particularly, CDR3 of the heavy chainmay constitute the most important determinants in antigen binding withinthe light and heavy chain variable regions. In some antibodies, theheavy chain CDR3 appears to constitute the major area of contact betweenthe antigen and the antibody. In vitro selection schemes in which CDR3alone is varied can be used to vary the binding properties of anantibody or determine which residues contribute to the binding of anantigen.

Naturally occurring antibodies typically include a signal sequence,which directs the antibody into the cellular pathway for proteinsecretion and which is typically not present in the mature antibody. Apolynucleotide encoding an antibody of the invention may encode anaturally occurring a signal sequence or a heterologous signal sequenceas described below.

In one embodiment, the antigen binding protein is an antibody comprisingfrom one to six of the exemplary CDRs described herein. The antibodiesof the invention may be of any type including IgM, IgG (including IgG1,IgG2, IgG3, IgG4), IgD, IgA, or IgE antibody. In a specific embodimentthe antigen binding protein is an IgG type antibody, e.g., a IgG1antibody.

In some embodiments, for example when the antigen binding protein is anantibody with complete heavy and light chains, the CDRs are all from thesame species, e.g., human. Alternatively, for example in embodimentswherein the antigen binding protein contains less than six CDRs from thesequences outlined above, additional CDRs may be either from otherspecies or may be different human CDRs than those depicted in theexemplary sequences. For example, HCDR3 and LCDR3 regions from theappropriate sequences identified herein may be used with HCDR1, HCDR2,LCDR1, and LCDR2 being optionally selected from alternate species ordifferent human antibody sequences, or combinations thereof. Forexample, the CDRs of the invention can replace the CDR regions ofcommercially relevant chimeric or humanized antibodies.

Specific embodiments utilize scaffold components of the antigen bindingproteins that are human components. In some embodiments, however, thescaffold components can be a mixture from different species. As such, ifthe antigen binding protein is an antibody, such antibody may be achimeric antibody and/or humanized antibody. In general, both “chimericantibodies” and humanized antibodies” refer to antibodies that combineregions from more than one species. For example, “chimeric antibodies”traditionally comprise variable region(s) from a mouse (or rat, in somecases) and the constant region(s) from a human.

“Humanized antibodies” generally refer to non-human antibodies that havehad the variable domain framework regions swapped for sequences found inhuman antibodies. Generally, in a humanized antibody, the entireantibody, except one or more CDRs, is encoded by a polynucleotide ofhuman origin or is identical to such an antibody except within one ormore CDRs. The CDRs, some or all of which are encoded by nucleic acidsoriginating in a non-human organism, are grafted into the beta-sheetframework of a human antibody variable region to create an antibody, thespecificity of which is determined by the engrafted CDRs. The creationof such antibodies is described in, e.g., WO 92/11018, Jones 1986,Nature 321:522-525, Verhoeyen et al., 1988, Science 239:1534-1536.Humanized antibodies can also be generated using mice with a geneticallyengineered immune system (Roque et al., 2004, Biotechnol. Prog.20:639-654). In the exemplary embodiments described herein, theidentified CDRs are human, and thus both humanized and chimericantibodies in this context include some non-human CDRs; for example,humanized antibodies may be generated that comprise the HCDR3 and LCDR3regions, with one or more of the other CDR regions being of a differentspecies origin.

In one embodiment, the OSMR antigen binding protein is a multispecificantibody, and notably a bispecfic antibody, also sometimes referred toas “diabodies.” These are antibodies that bind to two or more differentantigens or different epitopes on a single antigen. In certainembodiments, a bispecific antibody binds OSMR and an antigen on a humaneffector cell (e.g., T cell). Such antibodies are useful in targeting aneffector cell response against OSMR expressing cells, such as anOSMR-expressing tumor cell. In preferred embodiments, the human effectorcell antigen is CD3. U.S. Pat. No. 7,235,641. Methods of makingbispecific antibodies are known in the art. One such method involvesengineering the Fc portion of the heavy chains such as to create “knobs”and “holes” which facilitate heterodimer formation of the heavy chainswhen co-expressed in a cell. U.S. Pat. No. 7,695,963. Another methodalso involves engineering the Fc portion of the heavy chain but useselectrostatic steering to encourage heterodimer formation whilediscouraging homodimer formation of the heavy chains when co-expressedin a cell. WO 09/089,004, which is incorporated herein by reference inits entirety.

In one embodiment, the OSMR antigen binding protein is a minibody.Minibodies are minimized antibody-like proteins comprising a scFv joinedto a CH3 domain (Hu et al., 1996, Cancer Res. 56:3055-3061).

In one embodiment, the OSMR antigen binding protein is a domainantibody; see, for example U.S. Pat. No. 6,248,516. Domain antibodies(dAbs) are functional binding domains of antibodies, corresponding tothe variable regions of either the heavy (VH) or light (VL) chains ofhuman antibodies. dABs have a molecular weight of approximately 13 kDa,or less than one-tenth the size of a full antibody. dABs are wellexpressed in a variety of hosts including bacterial, yeast, andmammalian cell systems. In addition, dAbs are highly stable and retainactivity even after being subjected to harsh conditions, such asfreeze-drying or heat denaturation. See, for example, U.S. Pat. Nos.6,291,158; 6,582,915; 6,593,081; 6,172,197; US Serial No. 2004/0110941;European Patent 0368684; U.S. Pat. No. 6,696,245, WO04/058821,WO04/003019 and WO03/002609.

In one embodiment, the OSMR antigen binding protein is an antibodyfragment, that is a fragment of any of the antibodies outlined hereinthat retain binding specificity to OSMR. In various embodiments, theantibody binding proteins comprise, but are not limited to, a F(ab),F(ab′), F(ab′)2, Fv, or a single chain Fv fragments. At a minimum, anantibody, as meant herein, comprises a polypeptide that can bindspecifically to OSMR comprising all or part of a light or heavy chainvariable region, such as one or more CDRs.

Further examples of OSMR-binding antibody fragments include, but are notlimited to, (i) the Fab fragment consisting of VL, VH, CL and CH1domains, (ii) the Fd fragment consisting of the VH and CH1 domains,(iii) the Fv fragment consisting of the VL and VH domains of a singleantibody; (iv) the dAb fragment (Ward et al., 1989, Nature 341:544-546)which consists of a single variable, (v) isolated CDR regions, (vi)F(ab′)₂ fragments, a bivalent fragment comprising two linked Fabfragments (vii) single chain Fv molecules (scFv), wherein a VH domainand a VL domain are linked by a peptide linker which allows the twodomains to associate to form an antigen binding site (Bird et al., 1988,Science 242:423-426, Huston et al., 1988, Proc. Natl. Acad. Sci. U.S.A.85:5879-5883), (viii) bispecific single chain Fv dimers (PCT/US92/09965)and (ix) “diabodies” or “triabodies”, multivalent or multispecificfragments constructed by gene fusion (Tomlinson et. al., 2000, MethodsEnzymol. 326:461-479; WO94/13804; Holliger et al., 1993, Proc. Natl.Acad. Sci. U.S.A. 90:6444-6448). The antibody fragments may be modified.For example, the molecules may be stabilized by the incorporation ofdisulphide bridges linking the VH and VL domains (Reiter et al., 1996,Nature Biotech. 14:1239-1245). Aspects of the invention includeembodiments wherein the non-CDR components of these fragments are humansequences.

In one embodiment, the OSMR antigen binding protein is a fully humanantibody. In this embodiment, as outlined above, specific structurescomprise complete heavy and light chains depicted comprising the CDRregions. Additional embodiments utilize one or more of the CDRs of theinvention, with the other CDRs, framework regions, J and D regions,constant regions, etc., coming from other human antibodies. For example,the CDRs of the invention can replace the CDRs of any number of humanantibodies, particularly commercially relevant antibodies

Single chain antibodies may be formed by linking heavy and light chainvariable domain (Fv region) fragments via an amino acid bridge (shortpeptide linker), resulting in a single polypeptide chain. Suchsingle-chain Fvs (scFvs) have been prepared by fusing DNA encoding apeptide linker between DNAs encoding the two variable domainpolypeptides (V_(L) and V_(H)). The resulting polypeptides can fold backon themselves to form antigen-binding monomers, or they can formmultimers (e.g., dimers, trimers, or tetramers), depending on the lengthof a flexible linker between the two variable domains (Kortt et al.,1997, Prot. Eng. 10:423; Kortt et al., 2001, Biomol. Eng. 18:95-108). Bycombining different V_(L) and V_(H)-comprising polypeptides, one canform multimeric scFvs that bind to different epitopes (Kriangkum et al.,2001, Biomol. Eng. 18:31-40). Techniques developed for the production ofsingle chain antibodies include those described in U.S. Pat. No.4,946,778; Bird, 1988, Science 242:423; Huston et al., 1988, Proc. Natl.Acad. Sci. USA 85:5879; Ward et al., 1989, Nature 334:544, de Graaf etal., 2002, Methods Mol Biol. 178:379-87. Single chain antibodies derivedfrom antibodies provided herein (including but not limited to scFvscomprising the variable domain combinations of Ab1 LCv/Ab1 HCv (SEQ IDNO:27/SEQ ID NO:9), Ab2 LCv/Ab2 HCv (SEQ ID NO:28/SEQ ID NO:10), and Ab3LCv/Ab3 HCv (SEQ ID NO:29/SEQ ID NO:11), and combinations thereof areencompassed by the present invention. Exemplary single chain antibodiesinclude the following variable domain combinations: SEQ ID NO:27/SEQ IDNO:53; and SEQ ID NO:28/SEQ ID NO:54.

In one embodiment, the OSMR antigen binding protein is an antibodyfusion protein (sometimes referred to herein as an “antibodyconjugate”). The conjugate partner can be proteinaceous ornon-proteinaceous; the latter generally being generated using functionalgroups on the antigen binding protein and on the conjugate partner. Incertain embodiments, the antibody is conjugated to a non-proteinaceouschemical (drug) to form an antibody drug conjugate.

In one embodiment, the OSMR antigen binding protein is an antibodyanalog, sometimes referred to as “synthetic antibodies.” For example, avariety of work utilizes either alternative protein scaffolds orartificial scaffolds with grafted CDRs. Such scaffolds include, but arenot limited to, mutations introduced to stabilize the three-dimensionalstructure of the binding protein as well as wholly synthetic scaffoldsconsisting for example of biocompatible polymers. See, for example,Korndorfer et al., 2003, Proteins: Structure, Function, andBioinformatics, Volume 53, Issue 1:121-129. Roque et al., 2004,Biotechnol. Prog. 20:639-654. In addition, peptide antibody mimetics(“PAMs”) can be used, as well as work based on antibody mimeticsutilizing fibronection components as a scaffold.

By “protein,” as used herein, is meant at least two covalently attachedamino acids, which includes proteins, polypeptides, oligopeptides andpeptides. In some embodiments, the two or more covalently attached aminoacids are attached by a peptide bond. The protein may be made up ofnaturally occurring amino acids and peptide bonds, for example when theprotein is made recombinantly using expression systems and host cells,as outlined below. Alternatively, the protein may include syntheticamino acids (e.g., homophenylalanine, citrulline, ornithine, andnorleucine), or peptidomimetic structures, i.e., “peptide or proteinanalogs”, such as peptoids (see, Simon et al., 1992, Proc. Natl. Acad.Sci. U.S.A. 89:9367, incorporated by reference herein), which can beresistant to proteases or other physiological and/or storage conditions.Such synthetic amino acids may be incorporated in particular when theantigen binding protein is synthesized in vitro by conventional methodswell known in the art. In addition, any combination of peptidomimetic,synthetic and naturally occurring residues/structures can be used.“Amino acid” also includes imino acid residues such as proline andhydroxyproline. The amino acid “R group” or “side chain” may be ineither the (L)- or the (S)-configuration. In a specific embodiment, theamino acids are in the (L)- or (S)-configuration.

In certain aspects, the invention provides recombinant antigen bindingproteins that bind OSMR and, in some embodiments, a recombinant humanOSMR or portion thereof. In this context, a “recombinant protein” is aprotein made using recombinant techniques using any techniques andmethods known in the art, i.e., through the expression of a recombinantnucleic acid as described herein. Methods and techniques for theproduction of recombinant proteins are well known in the art.Embodiments of the invention include recombinant antigen bindingproteins that bind wild-type OSMR and variants thereof.

“Consisting essentially of” means that the amino acid sequence can varyby about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15% relativeto the recited SEQ ID NO: sequence and still retain biological activity,as described herein.

In some embodiments, the antigen binding proteins of the invention areisolated proteins or substantially pure proteins. An “isolated” proteinis unaccompanied by at least some of the material with which it isnormally associated in its natural state, for example constituting atleast about 5%, or at least about 50% by weight of the total protein ina given sample. It is understood that the isolated protein mayconstitute from 5 to 99.9% by weight of the total protein contentdepending on the circumstances. For example, the protein may be made ata significantly higher concentration through the use of an induciblepromoter or high expression promoter, such that the protein is made atincreased concentration levels. The definition includes the productionof an antigen binding protein in a wide variety of organisms and/or hostcells that are known in the art.

For amino acid sequences, sequence identity and/or similarity isdetermined by using standard techniques known in the art, including, butnot limited to, the local sequence identity algorithm of Smith andWaterman, 1981, Adv. Appl. Math. 2:482, the sequence identity alignmentalgorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, thesearch for similarity method of Pearson and Lipman, 1988, Proc. Nat.Acad. Sci. U.S.A. 85:2444, computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Drive, Madison,Wis.), the Best Fit sequence program described by Devereux et al., 1984,Nucl. Acid Res. 12:387-395, preferably using the default settings, or byinspection. Preferably, percent identity is calculated by FastDB basedupon the following parameters: mismatch penalty of 1; gap penalty of 1;gap size penalty of 0.33; and joining penalty of 30, “Current Methods inSequence Comparison and Analysis,” Macromolecule Sequencing andSynthesis, Selected Methods and Applications, pp 127-149 (1988), Alan R.Liss, Inc.

An example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments. It can also plot a tree showing the clusteringrelationships used to create the alignment. PILEUP uses a simplificationof the progressive alignment method of Feng & Doolittle, 1987, J. Mol.Evol. 35:351-360; the method is similar to that described by Higgins andSharp, 1989, CABIOS 5:151-153. Useful PILEUP parameters including adefault gap weight of 3.00, a default gap length weight of 0.10, andweighted end gaps.

Another example of a useful algorithm is the BLAST algorithm, describedin: Altschul et al., 1990, J. Mol. Biol. 215:403-410; Altschul et al.,1997, Nucleic Acids Res. 25:3389-3402; and Karin et al., 1993, Proc.Natl. Acad. Sci. U.S.A. 90:5873-5787. A particularly useful BLASTprogram is the WU-BLAST-2 program which was obtained from Altschul etal., 1996, Methods in Enzymology 266:460-480. WU-BLAST-2 uses severalsearch parameters, most of which are set to the default values. Theadjustable parameters are set with the following values: overlap span=1,overlap fraction=0.125, word threshold (T)=II. The HSP S and HSP S2parameters are dynamic values and are established by the program itselfdepending upon the composition of the particular sequence andcomposition of the particular database against which the sequence ofinterest is being searched; however, the values may be adjusted toincrease sensitivity.

An additional useful algorithm is gapped BLAST as reported by Altschulet al., 1993, Nucl. Acids Res. 25:3389-3402. Gapped BLAST uses BLOSUM-62substitution scores; threshold T parameter set to 9; the two-hit methodto trigger ungapped extensions, charges gap lengths of k a cost of 10+k;X_(u) set to 16, and X_(g) set to 40 for database search stage and to 67for the output stage of the algorithms. Gapped alignments are triggeredby a score corresponding to about 22 bits.

Generally, the amino acid homology, similarity, or identity betweenindividual variant CDRs are at least 80% to the sequences depictedherein, and more typically with preferably increasing homologies oridentities of at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, and almost 100%. In a similar manner, “percent (%) nucleic acidsequence identity” with respect to the nucleic acid sequence of thebinding proteins identified herein is defined as the percentage ofnucleotide residues in a candidate sequence that are identical with thenucleotide residues in the coding sequence of the antigen bindingprotein. A specific method utilizes the BLASTN module of WU-BLAST-2 setto the default parameters, with overlap span and overlap fraction set to1 and 0.125, respectively.

Generally, the nucleic acid sequence homology, similarity, or identitybetween the nucleotide sequences encoding individual variant CDRs andthe nucleotide sequences depicted herein are at least 80%, and moretypically with preferably increasing homologies or identities of atleast 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99%, and almost 100%.

Thus, a “variant CDR” is one with the specified homology, similarity, oridentity to the parent CDR of the invention, and shares biologicalfunction, including, but not limited to, at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% of the specificity and/or activity of the parent CDR.

While the site or region for introducing an amino acid sequencevariation is predetermined, the mutation per se need not bepredetermined. For example, in order to optimize the performance of amutation at a given site, random mutagenesis may be conducted at thetarget codon or region and the expressed antigen binding protein CDRvariants screened for the optimal combination of desired activity.Techniques for making substitution mutations at predetermined sites inDNA having a known sequence are well known, for example, M13 primermutagenesis and PCR mutagenesis. Screening of the mutants is done usingassays of antigen binding protein activities, such as OSMR binding.

Amino acid substitutions are typically of single residues; insertionsusually will be on the order of from about one (1) to about twenty (20)amino acid residues, although considerably larger insertions may betolerated. Deletions range from about one (1) to about twenty (20) aminoacid residues, although in some cases deletions may be much larger.

Substitutions, deletions, insertions or any combination thereof may beused to arrive at a final derivative or variant. Generally these changesare done on a few amino acids to minimize the alteration of themolecule, particularly the immunogenicity and specificity of the antigenbinding protein. However, larger changes may be tolerated in certaincircumstances. Conservative substitutions are generally made inaccordance with the following chart depicted as Table 3.

TABLE 3 Original Residue Exemplary Substitutions Ala Ser Arg Lys AsnGln, His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro His Asn, Gln Ile Leu,Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe Met, Leu, Tyr SerThr Thr Ser Trp Tyr Tyr Trp, Phe Val Ile, Leu

Substantial changes in function or immunological identity are made byselecting substitutions that are less conservative than those shown inTABLE 3. For example, substitutions may be made which more significantlyaffect: the structure of the polypeptide backbone in the area of thealteration, for example the alpha-helical or beta-sheet structure; thecharge or hydrophobicity of the molecule at the target site; or the bulkof the side chain. The substitutions which in general are expected toproduce the greatest changes in the polypeptide's properties are thosein which (a) a hydrophilic residue, e.g., seryl or threonyl, issubstituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl,phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substitutedfor (or by) any other residue; (c) a residue having an electropositiveside chain, e.g., lysyl, arginyl, or histidyl, is substituted for (orby) an electronegative residue, e.g., glutamyl or aspartyl; or (d) aresidue having a bulky side chain, e.g., phenylalanine, is substitutedfor (or by) one not having a side chain, e.g., glycine.

The variants typically exhibit the same qualitative biological activityand will elicit the same immune response as the naturally-occurringanalogue, although variants also are selected to modify thecharacteristics of the antigen binding protein proteins as needed.Alternatively, the variant may be designed such that the biologicalactivity of the antigen binding protein is altered. For example,glycosylation sites may be altered or removed as discussed herein.

Other derivatives of OSMR antibodies within the scope of this inventioninclude covalent or aggregative conjugates of OSMR antibodies, orfragments thereof, with other proteins or polypeptides, such as byexpression of recombinant fusion proteins comprising heterologouspolypeptides fused to the N-terminus or C-terminus of a OSMR antibodypolypeptide. For example, the conjugated peptide may be a heterologoussignal (or leader) polypeptide, e.g., the yeast alpha-factor leader, ora peptide such as an epitope tag. OSMR antibody-containing fusionproteins can comprise peptides added to facilitate purification oridentification of the OSMR antibody (e.g., poly-His). An OSMR antibodypolypeptide also can be linked to the FLAG peptide as described in Hoppet al., Bio/Technology 6:1204, 1988, and U.S. Pat. No. 5,011,912. TheFLAG peptide is highly antigenic and provides an epitope reversiblybound by a specific monoclonal antibody (mAb), enabling rapid assay andfacile purification of expressed recombinant protein. Reagents usefulfor preparing fusion proteins in which the FLAG peptide is fused to agiven polypeptide are commercially available (Sigma, St. Louis, Mo.).

In one embodiment, an oligomer is prepared using polypeptides derivedfrom immunoglobulins. Preparation of fusion proteins comprising certainheterologous polypeptides fused to various portions of antibody-derivedpolypeptides (including the Fc domain) has been described, e.g., byAshkenazi et al., 1991, PNAS USA 88:10535; Byrn et al., 1990, Nature344:677; and Hollenbaugh et al., 1992 “Construction of ImmunoglobulinFusion Proteins”, in Current Protocols in Immunology, Suppl. 4, pages10.19.1-10.19.11.

One embodiment of the present invention is directed to a dimercomprising two fusion proteins created by fusing an OSMR bindingfragment of an OSMR antibody to the Fc region of an antibody. The dimercan be made by, for example, inserting a gene fusion encoding the fusionprotein into an appropriate expression vector, expressing the genefusion in host cells transformed with the recombinant expression vector,and allowing the expressed fusion protein to assemble much like antibodymolecules, whereupon interchain disulfide bonds form between the Fcmoieties to yield the dimer.

The term “Fc polypeptide” as used herein includes native and muteinforms of polypeptides derived from the Fc region of an antibody.Truncated forms of such polypeptides containing the hinge region thatpromotes dimerization also are included. Fusion proteins comprising Fcmoieties (and oligomers formed therefrom) offer the advantage of facilepurification by affinity chromatography over Protein A or Protein Gcolumns.

One suitable Fc polypeptide, described in PCT application WO 93/10151(hereby incorporated by reference), is a single chain polypeptideextending from the N-terminal hinge region to the native C-terminus ofthe Fc region of a human IgG antibody. Another useful Fc polypeptide isthe Fc mutein described in U.S. Pat. No. 5,457,035 and in Baum et al.,1994, EMBO J. 13:3992-4001. The amino acid sequence of this mutein isidentical to that of the native Fc sequence presented in WO 93/10151,except that amino acid 19 has been changed from Leu to Ala, amino acid20 has been changed from Leu to Glu, and amino acid 22 has been changedfrom Gly to Ala. The mutein exhibits reduced affinity for Fc receptors.

In other embodiments, the variable portion of the heavy and/or lightchains of a OSMR antibody may be substituted for the variable portion ofan antibody heavy and/or light chain.

Another method for preparing oligomeric OSMR antibody derivativesinvolves use of a leucine zipper. Leucine zipper domains are peptidesthat promote oligomerization of the proteins in which they are found.Leucine zippers were originally identified in several DNA-bindingproteins (Landschulz et al., Science 240:1759-64, 1988), and have sincebeen found in a variety of different proteins. Among the known leucinezippers are naturally occurring peptides and derivatives thereof thatdimerize or trimerize. Examples of leucine zipper domains suitable forproducing soluble oligomeric proteins are described in PCT applicationWO 94/10308, and the leucine zipper derived from lung surfactant proteinD (SPD) described in Hoppe et al., 1994, FEBS Letters 344:191, herebyincorporated by reference. The use of a modified leucine zipper thatallows for stable trimerization of a heterologous protein fused theretois described in Fanslow et al., 1994, Semin. Immunol. 6:267-78. In oneapproach, recombinant fusion proteins comprising OSMR antibody fragmentor derivative fused to a leucine zipper peptide are expressed insuitable host cells, and the soluble oligomeric OSMR antibody fragmentsor derivatives that form are recovered from the culture supernatant.

Covalent modifications of antigen binding proteins are included withinthe scope of this invention, and are generally, but not always, donepost-translationally. For example, several types of covalentmodifications of the antigen binding protein are introduced into themolecule by reacting specific amino acid residues of the antigen bindingprotein with an organic derivatizing agent that is capable of reactingwith selected side chains or the N- or C-terminal residues.

Cysteinyl residues most commonly are reacted with α-haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residuesalso are derivatized by reaction with bromotrifluoroacetone,α-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylpyrocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1M sodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing alpha-amino-containing residues includeimidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;O-methylisourea; 2,4-pentanedione; and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high plc of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

The specific modification of tyrosyl residues may be made, withparticular interest in introducing spectral labels into tyrosyl residuesby reaction with aromatic diazonium compounds or tetranitromethane. Mostcommonly, N-acetylimidizole and tetranitromethane are used to formO-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosylresidues are iodinated using ¹²⁵I or ¹³¹I to prepare labeled proteinsfor use in radioimmunoassay, the chloramine T method described abovebeing suitable.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R′—N═C═N—R′), where R and R′ are optionallydifferent alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide.Furthermore, aspartyl and glutamyl residues are converted to asparaginyland glutaminyl residues by reaction with ammonium ions.

Derivatization with bifunctional agents is useful for crosslinkingantigen binding proteins to a water-insoluble support matrix or surfacefor use in a variety of methods. Commonly used crosslinking agentsinclude, e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidylpropionate), and bifunctional maleimidessuch as bis-N-maleimido-1,8-octane. Derivatizing agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide-activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537;and 4,330,440 are employed for protein immobilization.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues, respectively.Alternatively, these residues are deamidated under mildly acidicconditions. Either form of these residues falls within the scope of thisinvention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MolecularProperties, W. H. Freeman & Co., San Francisco, 1983, pp. 79-86),acetylation of the N-terminal amine, and amidation of any C-terminalcarboxyl group.

Another type of covalent modification of the antigen binding proteinincluded within the scope of this invention comprises altering theglycosylation pattern of the protein. As is known in the art,glycosylation patterns can depend on both the sequence of the protein(e.g., the presence or absence of particular glycosylation amino acidresidues, discussed below), or the host cell or organism in which theprotein is produced. Particular expression systems are discussed below.

Glycosylation of polypeptides is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tri-peptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tri-peptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid,most commonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antigen binding protein isconveniently accomplished by altering the amino acid sequence such thatit contains one or more of the above-described tri-peptide sequences(for N-linked glycosylation sites). The alteration may also be made bythe addition of, or substitution by, one or more serine or threonineresidues to the starting sequence (for O-linked glycosylation sites).For ease, the antigen binding protein amino acid sequence is preferablyaltered through changes at the DNA level, particularly by mutating theDNA encoding the target polypeptide at preselected bases such thatcodons are generated that will translate into the desired amino acids.

Another means of increasing the number of carbohydrate moieties on theantigen binding protein is by chemical or enzymatic coupling ofglycosides to the protein. These procedures are advantageous in thatthey do not require production of the protein in a host cell that hasglycosylation capabilities for N- and O-linked glycosylation. Dependingon the coupling mode used, the sugar(s) may be attached to (a) arginineand histidine, (b) free carboxyl groups, (c) free sulfhydryl groups suchas those of cysteine, (d) free hydroxyl groups such as those of serine,threonine, or hydroxyproline, (e) aromatic residues such as those ofphenylalanine, tyrosine, or tryptophan, or (f) the amide group ofglutamine. These methods are described in WO 87/05330 published Sep. 11,1987, and in Aplin and Wriston, 1981, CRC Crit. Rev. Biochem., pp.259-306.

Removal of carbohydrate moieties present on the starting antigen bindingprotein may be accomplished chemically or enzymatically. Chemicaldeglycosylation requires exposure of the protein to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving thepolypeptide intact. Chemical deglycosylation is described by Hakimuddinet al., 1987, Arch. Biochem. Biophys. 259:52 and by Edge et al., 1981,Anal. Biochem. 118:131. Enzymatic cleavage of carbohydrate moieties onpolypeptides can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al., 1987, Meth. Enzymol.138:350. Glycosylation at potential glycosylation sites may be preventedby the use of the compound tunicamycin as described by Duskin et al.,1982, J. Biol. Chem. 257:3105. Tunicamycin blocks the formation ofprotein-N-glycoside linkages.

Another type of covalent modification of the antigen binding proteincomprises linking the antigen binding protein to variousnonproteinaceous polymers, including, but not limited to, variouspolyols such as polyethylene glycol, polypropylene glycol orpolyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835;4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337. In addition, asis known in the art, amino acid substitutions may be made in variouspositions within the antigen binding protein to facilitate the additionof polymers such as PEG.

In some embodiments, the covalent modification of the antigen bindingproteins of the invention comprises the addition of one or more labels.

The term “labeling group” means any detectable label. Examples ofsuitable labeling groups include, but are not limited to, the following:radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc,¹¹¹In, ¹²⁵I, ¹³¹I), fluorescent groups (e.g., FITC, rhodamine,lanthanide phosphors), enzymatic groups (e.g., horseradish peroxidase,β-galactosidase, luciferase, alkaline phosphatase), chemiluminescentgroups, biotinyl groups, or predetermined polypeptide epitopesrecognized by a secondary reporter (e.g., leucine zipper pair sequences,binding sites for secondary antibodies, metal binding domains, epitopetags). In some embodiments, the labeling group is coupled to the antigenbinding protein via spacer arms of various lengths to reduce potentialsteric hindrance. Various methods for labeling proteins are known in theart and may be used in performing the present invention.

In general, labels fall into a variety of classes, depending on theassay in which they are to be detected: a) isotopic labels, which may beradioactive or heavy isotopes; b) magnetic labels (e.g., magneticparticles); c) redox active moieties; d) optical dyes; enzymatic groups(e.g. horseradish peroxidase, β-galactosidase, luciferase, alkalinephosphatase); e) biotinylated groups; and f) predetermined polypeptideepitopes recognized by a secondary reporter (e.g., leucine zipper pairsequences, binding sites for secondary antibodies, metal bindingdomains, epitope tags, etc.). In some embodiments, the labeling group iscoupled to the antigen binding protein via spacer arms of variouslengths to reduce potential steric hindrance. Various methods forlabeling proteins are known in the art and may be used in performing thepresent invention.

Specific labels include optical dyes, including, but not limited to,chromophores, phosphors and fluorophores, with the latter being specificin many instances. Fluorophores can be either “small molecule” fluores,or proteinaceous fluores.

By “fluorescent label” is meant any molecule that may be detected viaits inherent fluorescent properties. Suitable fluorescent labelsinclude, but are not limited to, fluorescein, rhodamine,tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins,pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade BlueJ, TexasRed, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy 5, Cy 5.5, LC Red 705,Oregon green, the Alexa-Fluor dyes (Alexa Fluor 350, Alexa Fluor 430,Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594,Alexa Fluor 633, Alexa Fluor 660, Alexa Fluor 680), Cascade Blue,Cascade Yellow and R-phycoerythrin (PE) (Molecular Probes, Eugene,Oreg.), FITC, Rhodamine, and Texas Red (Pierce, Rockford, Ill.), Cy5,Cy5.5, Cy7 (Amersham Life Science, Pittsburgh, Pa.). Suitable opticaldyes, including fluorophores, are described in Molecular Probes Handbookby Richard P. Haugland, hereby expressly incorporated by reference.

Suitable proteinaceous fluorescent labels also include, but are notlimited to, green fluorescent protein, including a Renilla, Ptilosarcus,or Aequorea species of GFP (Chalfie et al., 1994, Science 263:802-805),EGFP (Clontech Laboratories, Inc., Genbank Accession Number U55762),blue fluorescent protein (BFP, Quantum Biotechnologies, Inc. 1801 deMaisonneuve Blvd. West, 8th Floor, Montreal, Quebec, Canada H3H 1J9;Stauber, 1998, Biotechniques 24:462-471; Heim et al., 1996, Curr. Biol.6:178-182), enhanced yellow fluorescent protein (EYFP, ClontechLaboratories, Inc.), luciferase (Ichiki et al., 1993, J. Immunol.150:5408-5417), β galactosidase (Nolan et al., 1988, Proc. Natl. Acad.Sci. U.S.A. 85:2603-2607) and Renilla (WO92/15673, WO95/07463,WO98/14605, WO98/26277, WO99/49019, U.S. Pat. Nos. 5,292,658, 5,418,155,5,683,888, 5,741,668, 5,777,079, 5,804,387, 5,874,304, 5,876,995,5,925,558). All of the above-cited references are expressly incorporatedherein by reference.

The exemplary antigen binding proteins described herein have propertiesbased on the distinct epitope on OSMR bound by the antigen bindingprotein. The term “epitope” means the amino acids of a target moleculethat are contacted by an antigen binding protein, e.g., an antibody,when the antigen binding protein is bound to the target molecule. Anepitope can be contiguous or non-contiguous (e.g., (i) in a single-chainpolypeptide, amino acid residues that are not contiguous to one anotherin the polypeptide sequence but that within the context of the targetmolecule are bound by the antigen binding protein, or (ii) in amultimeric receptor comprising two or more individual components, e.g.,OSMR and gp130 or OSMR and IL-31 receptor A, amino acid residues arepresent on one or more of the individual components but are still boundby the antigen binding protein. Epitope determinants can includechemically active surface groupings of molecules such as amino acids,sugar side chains, phosphoryl or sulfonyl groups, and can have specificthree dimensional structural characteristics, and/or specific chargecharacteristics. Generally, antigen binding proteins specific for aparticular target molecule will preferentially recognize an epitope onthe target molecule in a complex mixture of proteins and/ormacromolecules.

Methods of characterizing the epitope bound by an antigen bindingprotein are well known in the art, including, but not limited to,binning (cross-competition) (Miller et al “Epitope binning of murinemonoclonal antibodies by a multiplexed pairing assay” J Immunol Methods(2011) 365, 118-25), peptide mapping (e.g., PEPSPOT™) (Albert et al “TheB-cell Epitope of the Monoclonal Anti-Factor VIII Antibody ESH8Characterized by Peptide Array Analysis” 2008 Thromb. Haemost. 99,634-7), mutagenesis methods such as chimeras (Song et al “EpitopeMapping of Ibalizumab, a Humanized Anti-CD4 Monoclonal Antibody withAnti-HIV-1 Activity in Infected Patients” J. Virol. (2010) 84,6935-6942), alanine scanning (Cunningham and Wells “High-resolutionepitope mapping of HGH-receptor interactions by alanine-scanningmutagenesis” Science (1989) 244, 1081-1085), arginine scanning (Lim etal “A diversity of antibody epitopes can induce signaling through theerythropoietin receptor” Biochemistry (2010) 49, 3797-3804), HD exchangemethods (Coates et al “Epitope mapping by amide hydrogen/deuteriumexchange coupled with immobilization of antibody, on-line proteolysis,liquid chromatography and mass spectrometry” Rapid Commun. MassSpectrom. (2009) 23 639-647), NMR cross saturation methods (Morgan et al“Precise epitope mapping of malaria parasite inhibitory antibodies byTROSY NMR cross-saturation” Biochemistry (2005) 44, 518-23), andcrystallography (Gerhardt et al “Structure of IL-17A in complex with apotent, fully human neutralizing antibody” J. Mol. Biol (2009) 394,905-21). The methods vary in the level of detail they provide as to theamino acids comprising the epitope. Example 4 provides an exemplarymethod of epitope binning.

Antigen binding proteins of the present invention include those thathave an overlapping epitope with an exemplary antigen binding proteindescribed herein, e.g., Ab1, Ab2, or Ab3. In certain embodiments, theantigen binding protein has an identical epitope as to the exemplaryantigen binding proteins. In other embodiments, the antigen bindingprotein binds only a subset of the same amino acids as the exemplaryantigen binding protein.

In certain embodiments, the OSMR antigen binding protein has anidentical or overlapping epitope as Ab1, Ab2, or Ab3, and comprises a) alight chain variable domain having at least 90% identity, at least 95%identity, or is identical to the amino acid sequence set forth in SEQ IDNO:27, SEQ ID NO:28, or SEQ ID NO:29; b) a heavy chain variable domainhaving at least 90% identity, at least 95% identity, or is identical tothe amino acid sequence set forth in SEQ ID NO:9, SEQ ID NO:10, or SEQID NO:11; or c) the light chain variable domain of a) and the heavychain variable domain of b).

In certain embodiments, the OSMR antigen binding protein has anidentical or overlapping epitope as Ab1, Ab2, or Ab3, and comprises alight chain variable domain having at least 90%, at least 95%, or isidentical to the amino acid sequence set forth in SEQ ID NO:27 and aheavy chain variable domain having at least 90%, at least 95%, or isidentical to the amino acid sequence set forth in SEQ ID NO:9; thosecomprising a light chain variable domain having at least 90%, at least95%, or is identical to the amino acid sequence set forth in SEQ IDNO:28 and a heavy chain variable domain having at least 90%, at least95%, or is identical to the amino acid sequence set forth in SEQ IDNO:10; and those comprising a light chain variable domain having atleast 90%, at least 95%, or is identical to the amino acid sequence setforth in SEQ ID NO:29 and a heavy chain variable domain having at least90%, at least 95%, or is identical to the amino acid sequence set forthin SEQ ID NO:11.

In certain embodiments, the OSMR antigen binding protein has anidentical or overlapping epitope as Ab1, Ab2, or Ab3, and comprises a) alight chain variable domain having no more than ten or no more than fiveamino acid additions, deletions or substitutions from the amino acidsequence set forth in SEQ ID NO:27, SEQ ID NO:28, or SEQ ID NO:29; b) aheavy chain variable domain having no more than ten or no more than fiveamino acid additions, deletions or substitutions from the amino acidsequence set forth in SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11; or c)the light chain variable domain of a) and the heavy chain variabledomain of b).

In certain embodiments, the OSMR antigen binding protein has anidentical or overlapping epitope as Ab1, Ab2, or Ab3, and comprises alight chain variable domain having no more than ten or no more than fiveamino acid additions, deletions or substitutions from the amino acidsequence set forth in SEQ ID NO:27 and a heavy chain variable domainhaving no more than ten or no more than five amino acid additions,deletions or substitutions from the amino acid sequence set forth in SEQID NO:9; those comprising a light chain variable domain having no morethan ten or no more than five amino acid additions, deletions orsubstitutions from the amino acid sequence set forth in SEQ ID NO:28 anda heavy chain variable domain having no more than ten or no more thanfive amino acid additions, deletions or substitutions from the aminoacid sequence set forth in SEQ ID NO:10; and those comprising a lightchain variable domain having no more than ten or no more than five aminoacid additions, deletions or substitutions from the amino acid sequenceset forth in SEQ ID NO:29 and a heavy chain variable domain having nomore than ten or no more than five amino acid additions, deletions orsubstitutions from the amino acid sequence set forth in SEQ ID NO:11.

An exemplary heavy chain variable domain variant of SEQ ID NO:9 containsan amino acid other than asparagine (for example, aspartic acid) at theposition corresponding to position 73 in SEQ ID NO:9. The amino acidsequence set forth in SEQ ID NO:53 is an example of a heavy chainvariable domain variant of SEQ ID NO:9.An exemplary heavy chain variable domain variant of SEQ ID NO:10contains an amino acid other than asparagine (for example, asparticacid) at the position corresponding to position 73 in SEQ ID NO:10. Theamino acid sequence set forth in SEQ ID NO:54 is an example of a heavychain variable domain variant of SEQ ID NO:10.

In certain embodiments, the OSMR antigen binding protein has anidentical or overlapping epitope as Ab1, Ab2, or Ab3, and comprises alight chain variable domain comprising a) an LCDR1 having no more thanthree amino acid additions, deletions, or substitutions from the LCDR1sequence set forth in SEQ ID NO:30; an LCDR2 having no more than threeamino acid additions, deletions, or substitutions from the LCDR2sequence set forth in SEQ ID NO:33; and an LCDR3 having no more thanthree amino acid additions, deletions, or substitutions from the LCDR3sequence set forth in SEQ ID NO:36; b) an LCDR1 having no more thanthree amino acid additions, deletions, or substitutions from the LCDR1sequence set forth in SEQ ID NO:31; an LCDR2 having no more than threeamino acid additions, deletions, or substitutions from the LCDR2sequence set forth in SEQ ID NO:34; and an LCDR3 having no more thanthree amino acid additions, deletions, or substitutions from the LCDR3sequence set forth in SEQ ID NO:37; or c) an LCDR1 having no more thanthree amino acid additions, deletions, or substitutions from the LCDR1sequence set forth in SEQ ID NO:32; an LCDR2 having no more than threeamino acid additions, deletions, or substitutions from the LCDR2sequence set forth in SEQ ID NO:35; and an LCDR3 having no more thanthree amino acid additions, deletions, or substitutions from the LCDR3sequence set forth in SEQ ID NO:38; and a heavy chain variable domaincomprising d) an HCDR1 having no more than three amino acid additions,deletions, or substitutions from the HCDR1 sequence set forth in SEQ IDNO:12; an HCDR2 having no more than three amino acid additions,deletions, or substitutions from the HCDR2 sequence set forth in SEQ IDNO:15; and an HCDR3 having no more than three amino acid additions,deletions, or substitutions from the HCDR3 sequence set forth in SEQ IDNO:18; e) an HCDR1 having no more than three amino acid additions,deletions, or substitutions from the HCDR1 sequence set forth in SEQ IDNO:13; an HCDR2 having no more than three amino acid additions,deletions, or substitutions from the HCDR2 sequence set forth in SEQ IDNO:16; and an HCDR3 having no more than three amino acid additions,deletions, or substitutions from the HCDR3 sequence set forth in SEQ IDNO:19; or f) an HCDR1 having no more than three amino acid additions,deletions, or substitutions from the HCDR1 sequence set forth in SEQ IDNO:14; an HCDR2 having no more than three amino acid additions,deletions, or substitutions from the HCDR2 sequence set forth in SEQ IDNO:17; and an HCDR3 having no more than three amino acid additions,deletions, or substitutions from the HCDR3 sequence set forth in SEQ IDNO:20.

Preferred OSMR antigen binding proteins described immediately aboveinclude those comprising the light chain variable domain of a) and theheavy chain variable domain of d); those comprising the light chainvariable domain of b) and the heavy chain variable domain of e); andthose comprising the light chain variable domain of c) and the heavychain variable domain of f).

OSMR antigen binding proteins comprising the light chain variable domainof a) and the heavy chain variable domain of d) can optionally contain aheavy chain variable domain that comprises an amino acid other thanasparagine (for example, aspartic acid) at the position corresponding toposition 73 in SEQ ID NO:9. In such embodiments, the heavy chainvariable domain optionally comprises the amino acid sequence set forthin SEQ ID NO:53.OSMR antigen binding proteins comprising the light chain variable domainof b) and the heavy chain variable domain of e) can optionally contain aheavy chain variable domain that comprises an amino acid other thanasparagine (for example, aspartic acid) at the position corresponding toposition 73 in SEQ ID NO:10. In such embodiments, the heavy chainvariable domain optionally comprises the amino acid sequence set forthin SEQ ID NO:54.

Antigen binding proteins that have an identical epitope or overlappingepitope will often cross-compete for binding to the antigen. Thus, incertain embodiments, an antigen binding protein of the inventioncross-competes with Ab1, Ab2, or Ab3. To “cross-compete” or“cross-competition” means the antigen binding proteins compete for thesame epitope or binding site on a target. Such competition can bedetermined by an assay in which the reference antigen binding protein(e.g., antibody or antigen-binding portion thereof) prevents or inhibitsspecific binding of a test antigen binding protein, and vice versa.Numerous types of competitive binding assays can be used to determine ifa test molecule competes with a reference molecule for binding. Examplesof assays that can be employed include solid phase direct or indirectradioimmunoassay (RIA), solid phase direct or indirect enzymeimmunoassay (EIA), sandwich competition assay (see, e.g., Stahli et al.(1983) Methods in Enzymology 9:242-253), solid phase directbiotin-avidin EIA (see, e.g., Kirkland et al., (1986) J. Immunol.137:3614-9), solid phase direct labeled assay, solid phase directlabeled sandwich assay, Luminex (Jia et al. “A novel method ofMultiplexed Competitive Antibody Binning for the characterization ofmonoclonal antibodies” J. Immunological Methods (2004) 288, 91-98) andsurface plasmon resonance (Song et al. “Epitope Mapping of Ibalizumab, aHumanized Anti-CD4 Monoclonal Antibody with Anti-HIV-1 Activity inInfected Patients” J. Virol. (2010) 84, 6935-42). An exemplary method ofdetermining cross-competition is described in Example 5. Usually, when acompeting antigen binding protein is present in excess, it will inhibitbinding of a reference antigen binding protein to a common antigen by atleast 50%, 55%, 60%, 65%, 70%, or 75%. In some instances, binding isinhibited by at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more.

Polynucleotides Encoding OSMR Antigen Binding Proteins

Encompassed within the invention are nucleic acids or isolated nucleicacids encoding OSMR antigen binding proteins, including antibodies, asdefined herein. Preferred nucleic acids include those that encode theexemplary light and heavy chains described herein.

An exemplary nucleic acid encoding Ab1 LC is a nucleic acid comprisingthe sequence set forth in SEQ ID NO:21.

An exemplary nucleic acid encoding Ab2 LC is a nucleic acid comprisingthe sequence set forth in SEQ ID NO:22.

An exemplary nucleic acid encoding Ab3 LC is a nucleic acid comprisingthe sequence set forth in SEQ ID NO:23.

An exemplary nucleic acid encoding Ab1 HC is a nucleic acid comprisingthe sequence set forth in SEQ ID NO:3.

An exemplary nucleic acid encoding Ab2 HC is a nucleic acid comprisingthe sequence set forth in SEQ ID NO:4.

An exemplary nucleic acid encoding Ab3 HC is a nucleic acid comprisingthe sequence set forth in SEQ ID NO:5.

An exemplary nucleic acid encoding a variant Ab1 HC is a nucleic acidcomprising the sequence set forth in SEQ ID NO:47.An exemplary nucleic acid encoding a variant Ab2 HC is a nucleic acidcomprising the sequence set forth in SEQ ID NO:48.An exemplary nucleic acid encoding a variant Ab3 HC is a nucleic acidcomprising the sequence set forth in SEQ ID NO:49.

Aspects of the invention include polynucleotide variants (e.g., due todegeneracy) that encode the amino acid sequences described herein.

Aspects of the invention include a variety of embodiments including, butnot limited to, the following exemplary embodiments.

An isolated nucleic acid comprising a polynucleotide, wherein saidpolynucleotide encodes one or more polypeptides comprising an amino acidsequence selected from the group consisting of:

A. 1. a light chain variable domain sequence that is at least 90%identical to a light chain variable domain sequence set forth in SEQ IDNOS:27-29;

2. a heavy chain variable domain sequence that is at least 90% identicalto a heavy chain variable domain sequence set forth in SEQ ID NOS:9-11;

3. a light chain variable domain of (1) and a heavy chain variabledomain of (2); and

B. a light chain variable domain comprising a CDR1, CDR2, CDR3 and/or aheavy chain variable domain comprising a CDR1, CDR2, CDR3 that are thesame or differ by no more than a total of three amino acid additions,substitutions, and/or deletions in each CDR from the followingsequences:

1. a light chain CDR1 (SEQ ID NO:30), CDR2 (SEQ ID NO:33), CDR3 (SEQ IDNO:36) or a heavy chain CDR1 (SEQ ID NO:12), CDR2 (SEQ ID NO:15), CDR3(SEQ ID NO:18) of Ab1;

2. a light chain CDR1 (SEQ ID NO:31), CDR2 (SEQ ID NO:34), CDR3 (SEQ IDNO:37) or a heavy chain CDR1 (SEQ ID NO:13), CDR2 (SEQ ID NO:16), CDR3(SEQ ID NO:19) of Ab2; and

3. a light chain CDR1 (SEQ ID NO:32), CDR2 (SEQ ID NO:35), CDR3 (SEQ IDNO:38) or a heavy chain CDR1 (SEQ ID NO:14), CDR2 (SEQ ID NO:17), CDR3(SEQ ID NO:20) of Ab3.

In some embodiments, the nucleic acid encodes a polypeptide thatcomprises the amino acid sequence set forth in SEQ ID NO:53 or SEQ IDNO:54.In some embodiments, the nucleic acid encodes a polypeptide thatcomprises the amino acid sequence set forth in SEQ ID NO:50, SEQ IDNO:51, or SEQ ID NO:52.

In preferred embodiments, the polypeptide encoded by the nucleic acid orisolated nucleic acid is a component of an antigen binding protein thatbinds OSMR.

Nucleotide sequences corresponding to the amino acid sequences describedherein, to be used as probes or primers for the isolation of nucleicacids or as query sequences for database searches, can be obtained by“back-translation” from the amino acid sequences, or by identificationof regions of amino acid identity with polypeptides for which the codingDNA sequence has been identified. The well-known polymerase chainreaction (PCR) procedure can be employed to isolate and amplify a DNAsequence encoding an OSMR antigen binding proteins or a desiredcombination of OSMR antigen binding protein polypeptide fragments.Oligonucleotides that define the desired termini of the combination ofDNA fragments are employed as 5′ and 3′ primers. The oligonucleotidescan additionally contain recognition sites for restrictionendonucleases, to facilitate insertion of the amplified combination ofDNA fragments into an expression vector. PCR techniques are described inSaiki et al., Science 239:487 (1988); Recombinant DNA Methodology, Wu etal., eds., Academic Press, Inc., San Diego (1989), pp. 189-196; and PCRProtocols: A Guide to Methods and Applications, Innis et. al., eds.,Academic Press, Inc. (1990).

Nucleic acid molecules of the invention include DNA and RNA in bothsingle-stranded and double-stranded form, as well as the correspondingcomplementary sequences. DNA includes, for example, cDNA, genomic DNA,chemically synthesized DNA, DNA amplified by PCR, and combinationsthereof. The nucleic acid molecules of the invention include full-lengthgenes or cDNA molecules as well as a combination of fragments thereof.The nucleic acids of the invention are preferentially derived from humansources, but the invention includes those derived from non-humanspecies, as well.

In some embodiments, nucleic acids of the invention are isolated nucleicacids. An “isolated nucleic acid” is a nucleic acid that has beenseparated from adjacent genetic sequences present in the genome of theorganism from which the nucleic acid was isolated, in the case ofnucleic acids isolated from naturally-occurring sources. In the case ofnucleic acids synthesized enzymatically from a template or chemically,such as PCR products, cDNA molecules, or oligonucleotides for example,it is understood that the nucleic acids resulting from such processesare isolated nucleic acids. An isolated nucleic acid molecule refers toa nucleic acid molecule in the form of a separate fragment or as acomponent of a larger nucleic acid construct. In one preferredembodiment, the nucleic acids are substantially free from contaminatingendogenous material. The nucleic acid molecule has preferably beenderived from DNA or RNA isolated at least once in substantially pureform and in a quantity or concentration enabling identification,manipulation, and recovery of its component nucleotide sequences bystandard biochemical methods (such as those outlined in Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1989)). Such sequences arepreferably provided and/or constructed in the form of an open readingframe uninterrupted by internal non-translated sequences, or introns,that are typically present in eukaryotic genes. Sequences ofnon-translated DNA can be present 5′ or 3′ from an open reading frame,where the same do not interfere with manipulation or expression of thecoding region.

The present invention also includes nucleic acids or isolated nucleicacids that hybridize under moderately stringent conditions, and morepreferably highly stringent conditions, to nucleic acids encoding OSMRantigen binding proteins as described herein. The basic parametersaffecting the choice of hybridization conditions and guidance fordevising suitable conditions are set forth by Sambrook, Fritsch, andManiatis (1989, Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and 11;and Current Protocols in Molecular Biology, 1995, Ausubel et al., eds.,John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4), and can be readilydetermined by those having ordinary skill in the art based on, forexample, the length and/or base composition of the DNA. One way ofachieving moderately stringent conditions involves the use of aprewashing solution containing 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0),hybridization buffer of about 50% formamide, 6×SSC, and a hybridizationtemperature of about 55 degrees C. (or other similar hybridizationsolutions, such as one containing about 50% formamide, with ahybridization temperature of about 42 degrees C.), and washingconditions of about 60 degrees C., in 0.5×SSC, 0.1% SDS. Generally,highly stringent conditions are defined as hybridization conditions asabove, but with washing at approximately 68 degrees C., 0.2×SSC, 0.1%SDS. SSPE (1×SSPE is 0.15M NaCl, 10 mM NaH.sub.2 PO.sub.4, and 1.25 mMEDTA, pH 7.4) can be substituted for SSC (1×SSC is 0.15M NaCl and 15 mMsodium citrate) in the hybridization and wash buffers; washes areperformed for 15 minutes after hybridization is complete. It should beunderstood that the wash temperature and wash salt concentration can beadjusted as necessary to achieve a desired degree of stringency byapplying the basic principles that govern hybridization reactions andduplex stability, as known to those skilled in the art and describedfurther below (see, e.g., Sambrook et al., 1989). When hybridizing anucleic acid to a target nucleic acid of unknown sequence, the hybridlength is assumed to be that of the hybridizing nucleic acid. Whennucleic acids of known sequence are hybridized, the hybrid length can bedetermined by aligning the sequences of the nucleic acids andidentifying the region or regions of optimal sequence complementarity.The hybridization temperature for hybrids anticipated to be less than 50base pairs in length should be 5 to 10.degrees C. less than the meltingtemperature (Tm) of the hybrid, where Tm is determined according to thefollowing equations. For hybrids less than 18 base pairs in length, Tm(degrees C.)=2(# of A+T bases)+4(# of #G+C bases). For hybrids above 18base pairs in length, Tm (degrees C.)=81.5+16.6(log₁₀ [Na⁺])+0.41(%G+C)−(600/N), where N is the number of bases in the hybrid, and [Na⁺] isthe concentration of sodium ions in the hybridization buffer ([Na⁺] for1×SSC=0.165M). Preferably, each such hybridizing nucleic acid has alength that is at least 15 nucleotides (or more preferably at least 18nucleotides, or at least 20 nucleotides, or at least 25 nucleotides, orat least 30 nucleotides, or at least 40 nucleotides, or most preferablyat least 50 nucleotides), or at least 25% (more preferably at least 50%,or at least 60%, or at least 70%, and most preferably at least 80%) ofthe length of the nucleic acid of the present invention to which ithybridizes, and has at least 60% sequence identity (more preferably atleast 70%, at least 75%, at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99%, and most preferably at least 99.5%) with thenucleic acid of the present invention to which it hybridizes, wheresequence identity is determined by comparing the sequences of thehybridizing nucleic acids when aligned so as to maximize overlap andidentity while minimizing sequence gaps as described in more detailabove.

The variants according to the invention are ordinarily prepared by sitespecific mutagenesis of nucleotides in the DNA encoding the antigenbinding protein, using cassette or PCR mutagenesis or other techniqueswell known in the art, to produce DNA encoding the variant, andthereafter expressing the recombinant DNA in cell culture as outlinedherein. However, antigen binding protein fragments comprising variantCDRs having up to about 100-150 residues may be prepared by in vitrosynthesis using established techniques. The variants typically exhibitthe same qualitative biological activity as the naturally occurringanalogue, e.g., binding to OSMR, although variants can also be selectedwhich have modified characteristics as will be more fully outlinedbelow.

As will be appreciated by those in the art, due to the degeneracy of thegenetic code, an extremely large number of nucleic acids may be made,all of which encode the CDRs (and heavy and light chains or othercomponents of the antigen binding protein) of the present invention.Thus, having identified a particular amino acid sequence, those skilledin the art could make any number of different nucleic acids, by simplymodifying the sequence of one or more codons in a way which does notchange the amino acid sequence of the encoded protein.

The present invention also provides expression systems and constructs inthe form of plasmids, expression vectors, transcription or expressioncassettes which comprise at least one polynucleotide as above. Inaddition, the invention provides host cells comprising such expressionsystems or constructs.

Typically, expression vectors used in any of the host cells will containsequences for plasmid maintenance and for cloning and expression ofexogenous nucleotide sequences. Such sequences, collectively referred toas “flanking sequences” in certain embodiments will typically includeone or more of the following nucleotide sequences: a promoter, one ormore enhancer sequences, an origin of replication, a transcriptionaltermination sequence, a complete intron sequence containing a donor andacceptor splice site, a sequence encoding a leader sequence forpolypeptide secretion, a ribosome binding site, a polyadenylationsequence, a polylinker region for inserting the nucleic acid encodingthe polypeptide to be expressed, and a selectable marker element. Eachof these sequences is discussed below.

Optionally, the vector may contain a “tag”-encoding sequence, i.e., anoligonucleotide molecule located at the 5′ or 3′ end of the OSMR antigenbinding protein coding sequence; the oligonucleotide sequence encodespolyHis (such as hexaHis), or another “tag” such as FLAG, HA(hemaglutinin influenza virus), or myc, for which commercially availableantibodies exist. This tag is typically fused to the polypeptide uponexpression of the polypeptide, and can serve as a means for affinitypurification or detection of the OSMR antigen binding protein from thehost cell. Affinity purification can be accomplished, for example, bycolumn chromatography using antibodies against the tag as an affinitymatrix. Optionally, the tag can subsequently be removed from thepurified OSMR antigen binding protein by various means such as usingcertain peptidases for cleavage.

Flanking sequences may be homologous (i.e., from the same species and/orstrain as the host cell), heterologous (i.e., from a species other thanthe host cell species or strain), hybrid (i.e., a combination offlanking sequences from more than one source), synthetic or native. Assuch, the source of a flanking sequence may be any prokaryotic oreukaryotic organism, any vertebrate or invertebrate organism, or anyplant, provided that the flanking sequence is functional in, and can beactivated by, the host cell machinery.

Flanking sequences useful in the vectors of this invention may beobtained by any of several methods well known in the art. Typically,flanking sequences useful herein will have been previously identified bymapping and/or by restriction endonuclease digestion and can thus beisolated from the proper tissue source using the appropriate restrictionendonucleases. In some cases, the full nucleotide sequence of a flankingsequence may be known. Here, the flanking sequence may be synthesizedusing the methods described herein for nucleic acid synthesis orcloning.

Whether all or only a portion of the flanking sequence is known, it maybe obtained using polymerase chain reaction (PCR) and/or by screening agenomic library with a suitable probe such as an oligonucleotide and/orflanking sequence fragment from the same or another species. Where theflanking sequence is not known, a fragment of DNA containing a flankingsequence may be isolated from a larger piece of DNA that may contain,for example, a coding sequence or even another gene or genes. Isolationmay be accomplished by restriction endonuclease digestion to produce theproper DNA fragment followed by isolation using agarose gelpurification, Qiagen® column chromatography (Chatsworth, Calif.), orother methods known to the skilled artisan. The selection of suitableenzymes to accomplish this purpose will be readily apparent to one ofordinary skill in the art.

An origin of replication is typically a part of those prokaryoticexpression vectors purchased commercially, and the origin aids in theamplification of the vector in a host cell. If the vector of choice doesnot contain an origin of replication site, one may be chemicallysynthesized based on a known sequence, and ligated into the vector. Forexample, the origin of replication from the plasmid pBR322 (New EnglandBiolabs, Beverly, Mass.) is suitable for most gram-negative bacteria,and various viral origins (e.g., SV40, polyoma, adenovirus, vesicularstomatitus virus (VSV), or papillomaviruses such as HPV or BPV) areuseful for cloning vectors in mammalian cells. Generally, the origin ofreplication component is not needed for mammalian expression vectors(for example, the SV40 origin is often used only because it alsocontains the virus early promoter).

A transcription termination sequence is typically located 3′ to the endof a polypeptide coding region and serves to terminate transcription.Usually, a transcription termination sequence in prokaryotic cells is aG-C rich fragment followed by a poly-T sequence. While the sequence iseasily cloned from a library or even purchased commercially as part of avector, it can also be readily synthesized using methods for nucleicacid synthesis such as those described herein.

A selectable marker gene encodes a protein necessary for the survivaland growth of a host cell grown in a selective culture medium. Typicalselection marker genes encode proteins that (a) confer resistance toantibiotics or other toxins, e.g., ampicillin, tetracycline, orkanamycin for prokaryotic host cells; (b) complement auxotrophicdeficiencies of the cell; or (c) supply critical nutrients not availablefrom complex or defined media. Specific selectable markers are thekanamycin resistance gene, the ampicillin resistance gene, and thetetracycline resistance gene. Advantageously, a neomycin resistance genemay also be used for selection in both prokaryotic and eukaryotic hostcells.

Other selectable genes may be used to amplify the gene that will beexpressed. Amplification is the process wherein genes that are requiredfor production of a protein critical for growth or cell survival arereiterated in tandem within the chromosomes of successive generations ofrecombinant cells. Examples of suitable selectable markers for mammaliancells include dihydrofolate reductase (DHFR) and promoterless thymidinekinase genes. Mammalian cell transformants are placed under selectionpressure wherein only the transformants are uniquely adapted to surviveby virtue of the selectable gene present in the vector. Selectionpressure is imposed by culturing the transformed cells under conditionsin which the concentration of selection agent in the medium issuccessively increased, thereby leading to the amplification of both theselectable gene and the DNA that encodes another gene, such as anantigen binding protein antibody that binds to OSMR polypeptide. As aresult, increased quantities of a polypeptide such as an OSMR antigenbinding protein are synthesized from the amplified DNA.

A ribosome-binding site is usually necessary for translation initiationof mRNA and is characterized by a Shine-Dalgarno sequence (prokaryotes)or a Kozak sequence (eukaryotes). The element is typically located 3′ tothe promoter and 5′ to the coding sequence of the polypeptide to beexpressed. In certain embodiments, one or more coding regions may beoperably linked to an internal ribosome binding site (IRES), allowingtranslation of two open reading frames from a single RNA transcript.

In some cases, such as where glycosylation is desired in a eukaryotichost cell expression system, one may manipulate the various pre- orprosequences to improve glycosylation or yield. For example, one mayalter the peptidase cleavage site of a particular signal peptide, or addprosequences, which also may affect glycosylation. The final proteinproduct may have, in the −1 position (relative to the first amino acidof the mature protein) one or more additional amino acids incident toexpression, which may not have been totally removed. For example, thefinal protein product may have one or two amino acid residues found inthe peptidase cleavage site, attached to the amino-terminus.Alternatively, use of some enzyme cleavage sites may result in aslightly truncated form of the desired polypeptide, if the enzyme cutsat such area within the mature polypeptide.

Expression and cloning vectors of the invention will typically contain apromoter that is recognized by the host organism and operably linked tothe molecule encoding the OSMR antigen binding protein. Promoters areuntranscribed sequences located upstream (i.e., 5′) to the start codonof a structural gene (generally within about 100 to 1000 bp) thatcontrol transcription of the structural gene. Promoters areconventionally grouped into one of two classes: inducible promoters andconstitutive promoters. Inducible promoters initiate increased levels oftranscription from DNA under their control in response to some change inculture conditions, such as the presence or absence of a nutrient or achange in temperature. Constitutive promoters, on the other hand,uniformly transcribe gene to which they are operably linked, that is,with little or no control over gene expression. A large number ofpromoters, recognized by a variety of potential host cells, are wellknown. A suitable promoter is operably linked to the DNA encoding heavychain or light chain comprising an OSMR antigen binding protein of theinvention by removing the promoter from the source DNA by restrictionenzyme digestion and inserting the desired promoter sequence into thevector.

Suitable promoters for use with yeast hosts are also well known in theart. Yeast enhancers are advantageously used with yeast promoters.Suitable promoters for use with mammalian host cells are well known andinclude, but are not limited to, those obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, retroviruses, hepatitis-B virus and most preferablySimian Virus 40 (SV40). Other suitable mammalian promoters includeheterologous mammalian promoters, for example, heat-shock promoters andthe actin promoter.

Additional promoters which may be of interest include, but are notlimited to: SV40 early promoter (Benoist et al., 1981, Nature290:304-310); CMV promoter (Thornsen et al., 1984, Proc. Natl. Acad.U.S.A. 81:659-663); the promoter contained in the 3′ long terminalrepeat of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22:787-797);herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad.Sci. U.S.A. 78:1444-1445); promoter and regulatory sequences from themetallothionine gene Prinster et al., 1982, Nature 296:39-42); andprokaryotic promoters such as the beta-lactamase promoter(Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. U.S.A.75:3727-3731); or the tac promoter (DeBoer et al., 1983, Proc. Natl.Acad. Sci. U.S.A. 80:21-25). Also of interest are the following animaltranscriptional control regions, which exhibit tissue specificity andhave been utilized in transgenic animals: the elastase I gene controlregion that is active in pancreatic acinar cells (Swift et al., 1984,Cell 38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant.Biol. 50:399-409; MacDonald, 1987, Hepatology 7:425-515); the insulingene control region that is active in pancreatic beta cells (Hanahan,1985, Nature 315:115-122); the immunoglobulin gene control region thatis active in lymphoid cells (Grosschedl et al., 1984, Cell 38:647-658;Adames et al., 1985, Nature 318:533-538; Alexander et al., 1987, Mol.Cell. Biol. 7:1436-1444); the mouse mammary tumor virus control regionthat is active in testicular, breast, lymphoid and mast cells (Leder etal., 1986, Cell 45:485-495); the albumin gene control region that isactive in liver (Pinkert et al., 1987, Genes and Devel. 1:268-276); thealpha-feto-protein gene control region that is active in liver (Krumlaufet al., 1985, Mol. Cell. Biol. 5:1639-1648; Hammer et al., 1987, Science253:53-58); the alpha 1-antitrypsin gene control region that is activein liver (Kelsey et al., 1987, Genes and Devel. 1:161-171); thebeta-globin gene control region that is active in myeloid cells (Mogramet al., 1985, Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94);the myelin basic protein gene control region that is active inoligodendrocyte cells in the brain (Readhead et al., 1987, Cell48:703-712); the myosin light chain-2 gene control region that is activein skeletal muscle (Sani, 1985, Nature 314:283-286); and thegonadotropic releasing hormone gene control region that is active in thehypothalamus (Mason et al., 1986, Science 234:1372-1378).

An enhancer sequence may be inserted into the vector to increasetranscription of DNA encoding light chain or heavy chain comprising anOSMR antigen binding protein of the invention by higher eukaryotes.Enhancers are cis-acting elements of DNA, usually about 10-300 bp inlength, that act on the promoter to increase transcription. Enhancersare relatively orientation and position independent, having been foundat positions both 5′ and 3′ to the transcription unit. Several enhancersequences available from mammalian genes are known (e.g., globin,elastase, albumin, alpha-feto-protein and insulin). Typically, however,an enhancer from a virus is used. The SV40 enhancer, the cytomegalovirusearly promoter enhancer, the polyoma enhancer, and adenovirus enhancersknown in the art are exemplary enhancing elements for the activation ofeukaryotic promoters. While an enhancer may be positioned in the vectoreither 5′ or 3′ to a coding sequence, it is typically located at a site5′ from the promoter. A sequence encoding an appropriate native orheterologous signal sequence (leader sequence or signal peptide) can beincorporated into an expression vector, to promote extracellularsecretion of the antibody. The choice of signal peptide or leaderdepends on the type of host cells in which the antibody is to beproduced, and a heterologous signal sequence can replace the nativesignal sequence. Examples of signal peptides that are functional inmammalian host cells include the following: the signal sequence forinterleukin-7 (IL-7) described in U.S. Pat. No. 4,965,195; the signalsequence for interleukin-2 receptor described in Cosman et al., 1984,Nature 312:768; the interleukin-4 receptor signal peptide described inEP Patent No. 0367 566; the type I interleukin-1 receptor signal peptidedescribed in U.S. Pat. No. 4,968,607; the type II interleukin-1 receptorsignal peptide described in EP Patent No. 0 460 846.

The vector may contain one or more elements that facilitate expressionwhen the vector is integrated into the host cell genome. Examplesinclude an EASE element (Aldrich et al. 2003 Biotechnol Prog.19:1433-38) and a matrix attachment region (MAR). MARs mediatestructural organization of the chromatin and may insulate the integratedvactor from “position” effect. Thus, MARs are particularly useful whenthe vector is used to create stable transfectants. A number of naturaland synthetic MAR-containing nucleic acids are known in the art, e.g.,U.S. Pat. Nos. 6,239,328; 7,326,567; 6,177,612; 6,388,066; 6,245,974;7,259,010; 6,037,525; 7,422,874; 7,129,062.

Expression vectors of the invention may be constructed from a startingvector such as a commercially available vector. Such vectors may or maynot contain all of the desired flanking sequences. Where one or more ofthe flanking sequences described herein are not already present in thevector, they may be individually obtained and ligated into the vector.Methods used for obtaining each of the flanking sequences are well knownto one skilled in the art.

After the vector has been constructed and a nucleic acid moleculeencoding light chain, a heavy chain, or a light chain and a heavy chaincomprising an OSMR antigen binding sequence has been inserted into theproper site of the vector, the completed vector may be inserted into asuitable host cell for amplification and/or polypeptide expression. Thetransformation of an expression vector for an OSMR antigen bindingprotein into a selected host cell may be accomplished by well knownmethods including transfection, infection, calcium phosphateco-precipitation, electroporation, microinjection, lipofection,DEAE-dextran mediated transfection, or other known techniques. Themethod selected will in part be a function of the type of host cell tobe used. These methods and other suitable methods are well known to theskilled artisan, and are set forth, for example, in Sambrook et al.,2001, supra.

A host cell, when cultured under appropriate conditions, synthesizes anOSMR antigen binding protein that can subsequently be collected from theculture medium (if the host cell secretes it into the medium) ordirectly from the host cell producing it (if it is not secreted). Theselection of an appropriate host cell will depend upon various factors,such as desired expression levels, polypeptide modifications that aredesirable or necessary for activity (such as glycosylation orphosphorylation) and ease of folding into a biologically activemolecule. A host cell may be eukaryotic or prokaryotic.

Mammalian cell lines available as hosts for expression are well known inthe art and include, but are not limited to, immortalized cell linesavailable from the American Type Culture Collection (ATCC) and any celllines used in an expression system known in the art can be used to makethe recombinant polypeptides of the invention. In general, host cellsare transformed with a recombinant expression vector that comprises DNAencoding a desired anti-OSMR antibody polypeptide. Among the host cellsthat may be employed are prokaryotes, yeast or higher eukaryotic cells.Prokaryotes include gram negative or gram positive organisms, forexample E. coli or bacilli. Higher eukaryotic cells include insect cellsand established cell lines of mammalian origin. Examples of suitablemammalian host cell lines include the COS-7 line of monkey kidney cells(ATCC CRL 1651) (Gluzman et al., 1981, Cell 23:175), L cells, 293 cells,C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells,or their derivatives such as Veggie CHO and related cell lines whichgrow in serum-free media (Rasmussen et al., 1998, Cytotechnology 28:31), HeLa cells, BHK (ATCC CRL 10) cell lines, and the CVI/EBNA cellline derived from the African green monkey kidney cell line CVI (ATCCCCL 70) as described by McMahan et al., 1991, EMBO J. 10: 2821, humanembryonic kidney cells such as 293, 293 EBNA or MSR 293, human epidermalA431 cells, human Colo205 cells, other transformed primate cell lines,normal diploid cells, cell strains derived from in vitro culture ofprimary tissue, primary explants, HL-60, U937, HaK or Jurkat cells.Optionally, mammalian cell lines such as HepG2/3B, KB, NIH 3T3 or S49,for example, can be used for expression of the polypeptide when it isdesirable to use the polypeptide in various signal transduction orreporter assays. Alternatively, it is possible to produce thepolypeptide in lower eukaryotes such as yeast or in prokaryotes such asbacteria. Suitable yeasts include Saccharomyces cerevisiae,Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeaststrain capable of expressing heterologous polypeptides. Suitablebacterial strains include Escherichia coli, Bacillus subtilis,Salmonella typhimurium, or any bacterial strain capable of expressingheterologous polypeptides. If the polypeptide is made in yeast orbacteria, it may be desirable to modify the polypeptide producedtherein, for example by phosphorylation or glycosylation of theappropriate sites, in order to obtain the functional polypeptide. Suchcovalent attachments can be accomplished using known chemical orenzymatic methods. The polypeptide can also be produced by operablylinking the nucleic acid or isolated nucleic acid of the invention tosuitable control sequences in one or more insect expression vectors, andemploying an insect expression system. Materials and methods forbaculovirus/insect cell expression systems are commercially available inkit form from, e.g., Invitrogen, San Diego, Calif., U.S.A. (the MaxBac®kit), and such methods are well known in the art, as described inSummers and Smith, Texas Agricultural Experiment Station Bulletin No.1555 (1987), and Luckow and Summers, Bio/Technology 6:47 (1988).Cell-free translation systems could also be employed to producepolypeptides using RNAs derived from nucleic acid constructs disclosedherein. Appropriate cloning and expression vectors for use withbacterial, fungal, yeast, and mammalian cellular hosts are described byPouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, NewYork, 1985). A host cell that comprises a nucleic acid or an isolatednucleic acid of the invention, preferably operably linked to at leastone expression control sequence, is a “recombinant host cell”.

In certain embodiments, cell lines may be selected through determiningwhich cell lines have high expression levels and constitutively produceantigen binding proteins with OSMR binding properties. In anotherembodiment, a cell line from the B cell lineage that does not make itsown antibody but has a capacity to make and secrete a heterologousantibody can be selected.

Cell-Depleting OSMR Antigen Binding Proteins

In preferred embodiments, the OSMR antigen binding protein binds OSMRand inhibits OSM and/or IL-31 binding, thereby reducing OSM- and/orIL-31-mediated signaling in OSMR-expressing cells. In certainembodiments, however, the OSMR antigen binding protein binds OSMR andtargets an OSMR-expressing cell for depletion. In various aspects, theOSMR antigen binding protein inhibits OSM and/or IL-31 binding andtargets the OSMR cell for depletion.

Cell-depleting OSMR antigen binding proteins are particularly useful fortreating diseases or disorders associated with over expression of OSMR,e.g., an autoimmune disease, inflammatory disease, a disease or disorderassociated with extracellular matrix deposition or remodeling, or anOSMR-expressing tumor. Methods of targeting cells with antigen bindingproteins, e.g. antibodies, are well known in the art. Exemplaryembodiments are discussed below.

Antibody Drug Conjugates

Embodiments of the invention include antibody drug conjugates (ADCs).Generally the ADC comprises an antibody conjugated to a chemotherapeuticagent, e.g., a cytotoxic agent, a cytostatic agent, a toxin, or aradioactive agent. A linker molecule can be used to conjugate the drugto the antibody. A wide variety of linkers and drugs useful in ADCtechnology are known in the art and may be used in embodiments of thepresent invention. (See US20090028856; US2009/0274713; US2007/0031402;WO2005/084390; WO2009/099728; U.S. Pat. No. 5,208,020; U.S. Pat. No.5,416,064; U.S. Pat. Nos. 5,475,092; 5,585,499; 6,436,931; 6,372,738;and 6,340,701, all incorporated herein by reference).

Linkers

In certain embodiments, the ADC comprises a linker made up of one ormore linker components. Exemplary linker components include6-maleimidocaproyl, maleimidopropanoyl, valine-citrulline,alanine-phenylalanine, p-aminobenzyloxycarbonyl, and those resultingfrom conjugation with linker reagents, including, but not limited to,N-succinimidyl 4-(2-pyridylthio) pentanoate (“SPP”), N-succinimidyl4-(N-maleimidomethyl) cyclohexane-1 carboxylate (“SMCC,” also referredto herein also as “MCC”), and N-succinimidyl (4-iodo-acetyl)aminobenzoate (“SIAB”).

Linkers may be a “cleavable” linker or a “non-cleavable” linker (Ducryand Stump, Bioconjugate Chem. 2010, 21, 5-13; incorporated herein byreference in its entirety) Cleavable linkers are designed to release thedrug when subjected to certain environment factors, e.g., wheninternalized into the target cell. Cleavable linkers include acid labilelinkers, protease sensitive linkers, photolabile linkers, dimethyllinker or disulfide-containing linkers. Non-cleavable linkers tend toremain covalently associated with at least one amino acid of theantibody and the drug upon internalization by and degradation within thetarget cell. An exemplary non-cleavable linker is MCC.

Drugs

In certain embodiments, the antibody is conjugated to a chemotherapeuticagent. Examples of chemotherapeutic agents include alkylating agents,such as thiotepa and cyclophosphamide (CYTOXAN™); alkyl sulfonates suchas busulfan, improsulfan and piposulfan; aziridines, such as benzodopa,carboquone, meturedopa, and uredopa; ethylenimines and methylamelaminesincluding altretamine, triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CBI-TMI); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,ranimustine; antibiotics, such as the enediyne antibiotics (e.g.calicheamicin, especially calicheamicin.gamma1 and calicheamicin thetaI, see, e.g., Angew Chem. Intl. Ed. Engl. 33:183-186 (1994); dynemicin,including dynemicin A; an esperamicin; as well as neocarzinostatinchromophore and related chromoprotein enediyne antibioticchromomophores), aclacinomysins, actinomycin, authramycin, azaserine,bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin;chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin (includingmorpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, nitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites, such as methotrexate and5-fluorouracil (5-FU); folic acid analogues, such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs, such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as, ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine,5-FU; androgens, such as calusterone, dromostanolone propionate,epitiostanol, mepitiostane, testolactone; anti-adrenals, such asaminoglutethimide, mitotane, trilostane; folic acid replenisher, such asfrolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;demecolcine; diaziquone; elfomithine; elliptinium acetate; anepothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan;lonidamine; maytansinoids, such as maytansine and ansamitocins;mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet;pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®;razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid;triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especiallyT-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine;dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids,e.g. paclitaxel (TAXOL™, Bristol-Myers Squibb Oncology, Princeton, N.J.)and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France);chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine;platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone;vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin;aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylomithine (DMFO); retinoic acid; capecitabine; andpharmaceutically acceptable salts, acids or derivatives of any of theabove. Also included in this definition are anti-hormonal agents thatact to regulate or inhibit hormone action on tumors, such asanti-estrogens including for example tamoxifen, raloxifene, aromataseinhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene,LY117018, onapristone, and toremifene (Fareston); and anti-androgens,such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin;siRNA and pharmaceutically acceptable salts, acids or derivatives of anyof the above. Other chemotherapeutic agents that can be used with thepresent invention are disclosed in US Publication No. 20080171040 or USPublication No. 20080305044, each of which is incorporated herein in itsentirety by reference.

It is contemplated that an antibody may be conjugated to two or moredifferent chemotherapeutic agents or a pharmaceutical composition maycomprise a mixture of antibodies wherein the antibody component isidentical except for being conjugated to a different chemotherapeuticagent. Such embodiments may be useful for targeting multiple biologicalpathways with a target cell.

In preferred embodiments, the ADC comprises an antibody conjugated toone or more maytansinoid molecules, which are mitotic inhibitors thatact by inhibiting tubulin polymerization. Maytansinoids, includingvarious modifications, are described in U.S. Pat. Nos. 3,896,111;4,151,042; 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814;4,294,757; 4,307,016; 4,308,268; 4,309,428; 4,313,946; 4,315,929;4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866; 4,424,219;4,450,254; 4,362,663; 4,371,533; and WO 2009/099728. Maytansinoid drugmoieties may be isolated from natural sources, produced usingrecombinant technology, or prepared synthetically. Exemplarymaytansinoids include C-19-dechloro (U.S. Pat. No. 4,256,746),C-20-hydroxy (or C-20-demethyl)+/−C-19-dechloro (U.S. Pat. Nos.4,307,016 and 4,361,650), C-20-demethoxy (or C-20-acyloxy (—OCOR),+/−dechrolo (U.S. Pat. No. 4,294,757), C-9-SH (U.S. Pat. No. 4,424,219),C-14-alkoxymethyl (demethoxy/CH2OR) (U.S. Pat. No. 4,331,598),C-14-hydroxymethyl or acyloxymethyl (CH2OH or CH2OAc) (U.S. Pat. No.4,450,254), C-15-hydroxy/acyloxy (U.S. Pat. No. 4,364,866), C-15-methoxy(U.S. Pat. Nos. 4,313,946 and 4,315,929), C-18-N-demethyl (U.S. Pat.Nos. 4,362,663 and 4,322,348), and 4,5-deoxy (U.S. Pat. No. 4,371,533).

Various positions on maytansinoid compounds may be used as the linkageposition, depending upon the type of link desired. For example, forforming an ester linkage, the C-3 position having a hydroxyl group, theC-14 position modified with hydrozymethyl, the C-15 position modifiedwith a hydroxyl a group, and the C-20 position having a hydroxyl groupare all suitable (U.S. Pat. Nos. 5,208,020, RE39151, and 6,913,748; USPatent Appl. Pub. Nos. 20060167245 and 20070037972, and WO 2009099728).

Preferred maytansinoids include those known in the art as DM1, DM3, andDM4 (US Pat. Appl. Pub. Nos. 2009030924 and 20050276812, incorporatedherein by reference).

ADCs containing maytansinoids, methods of making such ADCs, and theirtherapeutic use are disclosed in U.S. Pat. Nos. 5,208,020 and 5,416,064,US Pat. Appl. Pub. No. 20050276812, and WO 2009099728 (all incorporatedby reference herein). Linkers that are useful for making maytansinoidADCs are know in the art (U.S. Pat. No. 5,208,020 and US Pat. Appl. Pub.Nos. 2005016993 and 20090274713; all incorporated herein by reference).Maytansinoid ADCs comprising an SMCC linker may be prepared as disclosedin US Pat. Publ. No. 2005/0276812.

Effector Function-Enhanced Antibodies

One of the functions of the Fc portion of an antibody is to communicateto the immune system when the antibody binds its target. This isconsidered “effector function.” Communication leads toantibody-dependent cellular cytotoxicity (ADCC), antibody-dependentcellular phagocytosis (ADCP), and/or complement dependent cytotoxicity(CDC). ADCC and ADCP are mediated through the binding of the Fc to Fcreceptors on the surface of cells of the immune system. CDC is mediatedthrough the binding of the Fc with proteins of the complement system,e.g., C1q.

The IgG subclasses vary in their ability to mediate effector functions.For example, IgG1 is much superior to IgG2 and IgG4 at mediating ADCCand CDC. Thus, in embodiments wherein a cell expressing OSMR is targetedfor destruction, an anti-OSMR IgG1 antibody would be preferred.

The effector function of an antibody can be increased, or decreased, byintroducing one or more mutations into the Fc. Embodiments of theinvention include antigen binding proteins, e.g., antibodies, having anFc engineered to increase effector function (U.S. Pat. No. 7,317,091 andStrohl, Curr. Opin. Biotech., 20:685-691, 2009; both incorporated hereinby reference in its entirety). Exemplary IgG1 Fc molecules havingincreased effector function include (based on the Kabat numberingscheme) those have the following substitutions:

S239D/I332E

S239D/A330S/I332E

S239D/A330L/I332E

S298A/D333A/K334A

P247I/A339D

P247I/A339Q

D280H/K290S

D280H/K290S/S298D

D280H/K290S/S298V

F243L/R292P/Y300L

F243L/R292P/Y300L/P396L

F243L/R292P/Y300L/V305I/P396L

G236A/S239D/I332E

K326A/E333A

K326W/E333S

K290E/S298G/T299A

K290N/S298G/T299A

K290E/S298G/T299A/K326E

K290N/S298G/T299A/K326E

Further embodiments of the invention include antigen binding proteins,e.g., antibodies, having an Fc engineered to decrease effector function.Exemplary Fc molecules having decreased effector function include (basedon the Kabat numbering scheme) those have the following substitutions:

N297A (IgG1)

L234A/L235A (IgG1)

V234A/G237A (IgG2)

L235A/G237A/E318A (IgG4)

H268Q/V309L/A330S/A331S (IgG2)

C220S/C226S/C229S/P238S (IgG1)

C226S/C229S/E233P/L234V/L235A (IgG1)

L234F/L235E/P331S (IgG1)

S267E/L328F (IgG1)

Another method of increasing effector function of IgG Fc-containingproteins is by reducing the fucosylation of the Fc. Removal of the corefucose from the biantennary complex-type oligosachharides attached tothe Fc greatly increased ADCC effector function without altering antigenbinding or CDC effector function. Several ways are known for reducing orabolishing fucosylation of Fc-containing molecules, e.g., antibodies.These include recombinant expression in certain mammalian cell linesincluding a FUT8 knockout cell line, variant CHO line Lec13, rathybridoma cell line YB2/0, a cell line comprising a small interferingRNA specifically against the FUT8 gene, and a cell line coexpressingacetylglucosaminyltransferase III and Golgi α-mannosidase II.Alternatively, the Fc-containing molecule may be expressed in anon-mammalian cell such as a plant cell, yeast, or prokaryotic cell,e.g., E. coli. Thus, in certain embodiments of the invention, acomposition comprises an antibody, e.g., Ab1, Ab2, or Ab3 having reducedfucosylation or lacking fucosylation altogether.

Pharmaceutical Compositions

In some embodiments, the invention provides a pharmaceutical compositioncomprising a therapeutically effective amount of one or a plurality ofthe antigen binding proteins of the invention together with apharmaceutically effective diluents, carrier, solubilizer, emulsifier,preservative, and/or adjuvant. In certain embodiments, the antigenbinding protein is an antibody. Pharmaceutical compositions of theinvention include, but are not limited to, liquid, frozen, andlyophilized compositions.

Preferably, formulation materials are nontoxic to recipients at thedosages and concentrations employed. In specific embodiments,pharmaceutical compositions comprising a therapeutically effectiveamount of an OSMR antigen binding protein, e.g., an OSMR-bindingantibody, are provided.

In certain embodiments, the pharmaceutical composition may containformulation materials for modifying, maintaining or preserving, forexample, the pH, osmolarity, viscosity, clarity, color, isotonicity,odor, sterility, stability, rate of dissolution or release, adsorptionor penetration of the composition. In such embodiments, suitableformulation materials include, but are not limited to, amino acids (suchas glycine, glutamine, asparagine, arginine, proline, or lysine);antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite orsodium hydrogen-sulfite); buffers (such as borate, bicarbonate,Tris-HCl, citrates, phosphates or other organic acids); bulking agents(such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine,polyvinylpyrrolidone, beta-cyclodextrin orhydroxypropyl-beta-cyclodextrin); fillers; monosaccharides;disaccharides; and other carbohydrates (such as glucose, mannose ordextrins); proteins (such as serum albumin, gelatin or immunoglobulins);coloring, flavoring and diluting agents; emulsifying agents; hydrophilicpolymers (such as polyvinylpyrrolidone); low molecular weightpolypeptides; salt-forming counterions (such as sodium); preservatives(such as benzalkonium chloride, benzoic acid, salicylic acid,thimerosal, phenethyl alcohol, methylparaben, propylparaben,chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such asglycerin, propylene glycol or polyethylene glycol); sugar alcohols (suchas mannitol or sorbitol); suspending agents; surfactants or wettingagents (such as pluronics, PEG, sorbitan esters, polysorbates such aspolysorbate 20, polysorbate, triton, tromethamine, lecithin,cholesterol, tyloxapal); stability enhancing agents (such as sucrose orsorbitol); tonicity enhancing agents (such as alkali metal halides,preferably sodium or potassium chloride, mannitol sorbitol); deliveryvehicles; diluents; excipients and/or pharmaceutical adjuvants. See,REMINGTON'S PHARMACEUTICAL SCIENCES, 18″ Edition, (A. R. Genrmo, ed.),1990, Mack Publishing Company.

In certain embodiments, the optimal pharmaceutical composition will bedetermined by one skilled in the art depending upon, for example, theintended route of administration, delivery format and desired dosage.See, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, supra. In certainembodiments, such compositions may influence the physical state,stability, rate of in vivo release and rate of in vivo clearance of theantigen binding proteins of the invention. In certain embodiments, theprimary vehicle or carrier in a pharmaceutical composition may be eitheraqueous or non-aqueous in nature. For example, a suitable vehicle orcarrier may be water for injection, physiological saline solution orartificial cerebrospinal fluid, possibly supplemented with othermaterials common in compositions for parenteral administration. Neutralbuffered saline or saline mixed with serum albumin are further exemplaryvehicles. In specific embodiments, pharmaceutical compositions compriseTris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5,and may further include sorbitol or a suitable substitute therefor. Incertain embodiments of the invention, OSMR antigen binding proteincompositions may be prepared for storage by mixing the selectedcomposition having the desired degree of purity with optionalformulation agents (REMINGTON'S PHARMACEUTICAL SCIENCES, supra) in theform of a lyophilized cake or an aqueous solution. Further, in certainembodiments, the OSMR antigen binding protein product may be formulatedas a lyophilizate using appropriate excipients such as sucrose.

The pharmaceutical compositions of the invention can be selected forparenteral delivery. Alternatively, the compositions may be selected forinhalation or for delivery through the digestive tract, such as orally.Preparation of such pharmaceutically acceptable compositions is withinthe skill of the art. The formulation components are present preferablyin concentrations that are acceptable to the site of administration. Incertain embodiments, buffers are used to maintain the composition atphysiological pH or at a slightly lower pH, typically within a pH rangeof from about 5 to about 8.

When parenteral administration is contemplated, the therapeuticcompositions for use in this invention may be provided in the form of apyrogen-free, parenterally acceptable aqueous solution comprising thedesired OSMR antigen binding protein in a pharmaceutically acceptablevehicle. A particularly suitable vehicle for parenteral injection issterile distilled water in which the OSMR antigen binding protein isformulated as a sterile, isotonic solution, properly preserved. Incertain embodiments, the preparation can involve the formulation of thedesired molecule with an agent, such as injectable microspheres,bio-erodible particles, polymeric compounds (such as polylactic acid orpolyglycolic acid), beads or liposomes, that may provide controlled orsustained release of the product which can be delivered via depotinjection. In certain embodiments, hyaluronic acid may also be used,having the effect of promoting sustained duration in the circulation. Incertain embodiments, implantable drug delivery devices may be used tointroduce the desired antigen binding protein.

Pharmaceutical compositions of the invention can be formulated forinhalation. In these embodiments, OSMR antigen binding proteins areadvantageously formulated as a dry, inhalable powder. In specificembodiments, OSMR antigen binding protein inhalation solutions may alsobe formulated with a propellant for aerosol delivery. In certainembodiments, solutions may be nebulized. Pulmonary administration andformulation methods therefore are further described in InternationalPatent Application No. PCT/US94/001875, which is incorporated byreference and describes pulmonary delivery of chemically modifiedproteins.

It is also contemplated that formulations can be administered orally.OSMR antigen binding proteins that are administered in this fashion canbe formulated with or without carriers customarily used in thecompounding of solid dosage forms such as tablets and capsules. Incertain embodiments, a capsule may be designed to release the activeportion of the formulation at the point in the gastrointestinal tractwhen bioavailability is maximized and pre-systemic degradation isminimized Additional agents can be included to facilitate absorption ofthe OSMR antigen binding protein. Diluents, flavorings, low meltingpoint waxes, vegetable oils, lubricants, suspending agents, tabletdisintegrating agents, and binders may also be employed.

Additional pharmaceutical compositions will be evident to those skilledin the art, including formulations involving OSMR antigen bindingproteins in sustained- or controlled-delivery formulations. Techniquesfor formulating a variety of other sustained- or controlled-deliverymeans, such as liposome carriers, bio-erodible microparticles or porousbeads and depot injections, are also known to those skilled in the art.See, for example, International Patent Application No. PCT/US93/00829,which is incorporated by reference and describes controlled release ofporous polymeric microparticles for delivery of pharmaceuticalcompositions. Sustained-release preparations may include semipermeablepolymer matrices in the form of shaped articles, e.g., films, ormicrocapsules. Sustained release matrices may include polyesters,hydrogels, polylactides (as disclosed in U.S. Pat. No. 3,773,919 andEuropean Patent Application Publication No. EP 058481, each of which isincorporated by reference), copolymers of L-glutamic acid and gammaethyl-L-glutamate (Sidman et al., 1983, Biopolymers 2:547-556), poly(2-hydroxyethyl-methacrylate) (Langer et al., 1981, J. Biomed. Mater.Res. 15:167-277 and Langer, 1982, Chem. Tech. 12:98-105), ethylene vinylacetate (Langer et al., 1981, supra) or poly-D(−)-3-hydroxybutyric acid(European Patent Application Publication No. EP 133,988). Sustainedrelease compositions may also include liposomes that can be prepared byany of several methods known in the art. See, e.g., Eppstein et al.,1985, Proc. Natl. Acad. Sci. U.S.A. 82:3688-3692; European PatentApplication Publication Nos. EP 036,676; EP 088,046 and EP 143,949,incorporated by reference.

Pharmaceutical compositions used for in vivo administration aretypically provided as sterile preparations. Sterilization can beaccomplished by filtration through sterile filtration membranes. Whenthe composition is lyophilized, sterilization using this method may beconducted either prior to or following lyophilization andreconstitution. Compositions for parenteral administration can be storedin lyophilized form or in a solution. Parenteral compositions generallyare placed into a container having a sterile access port, for example,an intravenous solution bag or vial having a stopper pierceable by ahypodermic injection needle.

Aspects of the invention includes self-buffering OSMR antigen bindingprotein formulations, which can be used as pharmaceutical compositions,as described in international patent application WO 06138181A2(PCT/US2006/022599), which is incorporated by reference in its entiretyherein.

As discussed above, certain embodiments provide OSMR antigen bindingproteins protein compositions, particularly pharmaceutical OSMR antigenbinding protein compositions that comprise, in addition to the OSMRantigen binding protein, one or more excipients such as thoseillustratively described in this section and elsewhere herein.Excipients can be used in the invention in this regard for a widevariety of purposes, such as adjusting physical, chemical, or biologicalproperties of formulations, such as adjustment of viscosity, and orprocesses of the invention to improve effectiveness and or to stabilizesuch formulations and processes against degradation and spoilage due to,for instance, stresses that occur during manufacturing, shipping,storage, pre-use preparation, administration, and thereafter.

A variety of expositions are available on protein stabilization andformulation materials and methods useful in this regard, such as Arakawaet al., “Solvent interactions in pharmaceutical formulations,” PharmRes. 8(3): 285-91 (1991); Kendrick et al., “Physical stabilization ofproteins in aqueous solution,” in: RATIONAL DESIGN OF STABLE PROTEINFORMULATIONS: THEORY AND PRACTICE, Carpenter and Manning, eds.Pharmaceutical Biotechnology. 13: 61-84 (2002), and Randolph et al.,“Surfactant-protein interactions,” Pharm Biotechnol. 13: 159-75 (2002),each of which is herein incorporated by reference in its entirety,particularly in parts pertinent to excipients and processes of the samefor self-buffering protein formulations in accordance with the currentinvention, especially as to protein pharmaceutical products andprocesses for veterinary and/or human medical uses.

Salts may be used in accordance with certain embodiments of theinvention to, for example, adjust the ionic strength and/or theisotonicity of a formulation and/or to improve the solubility and/orphysical stability of a protein or other ingredient of a composition inaccordance with the invention.

As is well known, ions can stabilize the native state of proteins bybinding to charged residues on the protein's surface and by shieldingcharged and polar groups in the protein and reducing the strength oftheir electrostatic interactions, attractive, and repulsiveinteractions. Ions also can stabilize the denatured state of a proteinby binding to, in particular, the denatured peptide linkages (—CONH) ofthe protein. Furthermore, ionic interaction with charged and polargroups in a protein also can reduce intermolecular electrostaticinteractions and, thereby, prevent or reduce protein aggregation andinsolubility.

Ionic species differ significantly in their effects on proteins. Anumber of categorical rankings of ions and their effects on proteinshave been developed that can be used in formulating pharmaceuticalcompositions in accordance with the invention. One example is theHofmeister series, which ranks ionic and polar non-ionic solutes bytheir effect on the conformational stability of proteins in solution.Stabilizing solutes are referred to as “kosmotropic.” Destabilizingsolutes are referred to as “chaotropic.” Kosmotropes commonly are usedat high concentrations (e.g., >1 molar ammonium sulfate) to precipitateproteins from solution (“salting-out”). Chaotropes commonly are used todenture and/or to solubilize proteins (“salting-in”). The relativeeffectiveness of ions to “salt-in” and “salt-out” defines their positionin the Hofmeister series.

Free amino acids can be used in OSMR antigen binding proteinformulations in accordance with various embodiments of the invention asbulking agents, stabilizers, and antioxidants, as well as other standarduses. Lysine, proline, serine, and alanine can be used for stabilizingproteins in a formulation. Glycine is useful in lyophilization to ensurecorrect cake structure and properties. Arginine may be useful to inhibitprotein aggregation, in both liquid and lyophilized formulations.Methionine is useful as an antioxidant.

Polyols include sugars, e.g. mannitol, sucrose, and sorbitol andpolyhydric alcohols such as, for instance, glycerol and propyleneglycol, and, for purposes of discussion herein, polyethylene glycol(PEG) and related substances. Polyols are kosmotropic. They are usefulstabilizing agents in both liquid and lyophilized formulations toprotect proteins from physical and chemical degradation processes.Polyols also are useful for adjusting the tonicity of formulations.

Among polyols useful in select embodiments of the invention is mannitol,commonly used to ensure structural stability of the cake in lyophilizedformulations. It ensures structural stability to the cake. It isgenerally used with a lyoprotectant, e.g., sucrose. Sorbitol and sucroseare among preferred agents for adjusting tonicity and as stabilizers toprotect against freeze-thaw stresses during transport or the preparationof bulks during the manufacturing process. Reducing sugars (whichcontain free aldehyde or ketone groups), such as glucose and lactose,can glycate surface lysine and arginine residues. Therefore, theygenerally are not among preferred polyols for use in accordance with theinvention. In addition, sugars that form such reactive species, such assucrose, which is hydrolyzed to fructose and glucose under acidicconditions, and consequently engenders glycation, also is not amongpreferred polyols of the invention in this regard. PEG is useful tostabilize proteins and as a cryoprotectant and can be used in theinvention in this regard.

Embodiments of the OSMR antigen binding protein formulations furthercomprise surfactants. Protein molecules may be susceptible to adsorptionon surfaces and to denaturation and consequent aggregation atair-liquid, solid-liquid, and liquid-liquid interfaces. These effectsgenerally scale inversely with protein concentration. These deleteriousinteractions generally scale inversely with protein concentration andtypically are exacerbated by physical agitation, such as that generatedduring the shipping and handling of a product.

Surfactants routinely are used to prevent, minimize, or reduce surfaceadsorption. Useful surfactants in the invention in this regard includepolysorbate 20, polysorbate 80, other fatty acid esters of sorbitanpolyethoxylates, and poloxamer 188.

Surfactants also are commonly used to control protein conformationalstability. The use of surfactants in this regard is protein-specificsince, any given surfactant typically will stabilize some proteins anddestabilize others.

Polysorbates are susceptible to oxidative degradation and often, assupplied, contain sufficient quantities of peroxides to cause oxidationof protein residue side-chains, especially methionine. Consequently,polysorbates should be used carefully, and when used, should be employedat their lowest effective concentration. In this regard, polysorbatesexemplify the general rule that excipients should be used in theirlowest effective concentrations.

Embodiments of OSMR antigen binding protein formulations furthercomprise one or more antioxidants. To some extent deleterious oxidationof proteins can be prevented in pharmaceutical formulations bymaintaining proper levels of ambient oxygen and temperature and byavoiding exposure to light. Antioxidant excipients can be used as wellto prevent oxidative degradation of proteins. Among useful antioxidantsin this regard are reducing agents, oxygen/free-radical scavengers, andchelating agents. Antioxidants for use in therapeutic proteinformulations in accordance with the invention preferably arewater-soluble and maintain their activity throughout the shelf life of aproduct. EDTA is a preferred antioxidant in accordance with theinvention in this regard.

Antioxidants can damage proteins. For instance, reducing agents, such asglutathione in particular, can disrupt intramolecular disulfidelinkages. Thus, antioxidants for use in the invention are selected to,among other things, eliminate or sufficiently reduce the possibility ofthemselves damaging proteins in the formulation.

Formulations in accordance with the invention may include metal ionsthat are protein co-factors and that are necessary to form proteincoordination complexes, such as zinc necessary to form certain insulinsuspensions. Metal ions also can inhibit some processes that degradeproteins. However, metal ions also catalyze physical and chemicalprocesses that degrade proteins.

Magnesium ions (10-120 mM) can be used to inhibit isomerization ofaspartic acid to isoaspartic acid. Ca⁺² ions (up to 100 mM) can increasethe stability of human deoxyribonuclease. Mg⁺², Mn⁺², and Zn⁺², however,can destabilize rhDNase. Similarly, Ca⁺² and Sr⁺² can stabilize FactorVIII, it can be destabilized by Mg⁺², Mn⁺² and Zn⁺², Cu⁺² and Fe⁺², andits aggregation can be increased by Al⁺³ ions.

Embodiments of the OSMR antigen binding protein formulations furthercomprise one or more preservatives. Preservatives are necessary whendeveloping multi-dose parenteral formulations that involve more than oneextraction from the same container. Their primary function is to inhibitmicrobial growth and ensure product sterility throughout the shelf-lifeor term of use of the drug product. Commonly used preservatives includebenzyl alcohol, phenol and m-cresol. Although preservatives have a longhistory of use with small-molecule parenterals, the development ofprotein formulations that includes preservatives can be challenging.Preservatives almost always have a destabilizing effect (aggregation) onproteins, and this has become a major factor in limiting their use inmulti-dose protein formulations. To date, most protein drugs have beenformulated for single-use only. However, when multi-dose formulationsare possible, they have the added advantage of enabling patientconvenience, and increased marketability. A good example is that ofhuman growth hormone (hGH) where the development of preservedformulations has led to commercialization of more convenient, multi-useinjection pen presentations. At least four such pen devices containingpreserved formulations of hGH are currently available on the market.Norditropin (liquid, Novo Nordisk), Nutropin AQ (liquid, Genentech) &Genotropin (lyophilized—dual chamber cartridge, Pharmacia & Upjohn)contain phenol while Somatrope (Eli Lilly) is formulated with m-cresol.

Several aspects need to be considered during the formulation anddevelopment of preserved dosage forms. The effective preservativeconcentration in the drug product must be optimized. This requirestesting a given preservative in the dosage form with concentrationranges that confer anti-microbial effectiveness without compromisingprotein stability.

As might be expected, development of liquid formulations containingpreservatives are more challenging than lyophilized formulations.Freeze-dried products can be lyophilized without the preservative andreconstituted with a preservative containing diluent at the time of use.This shortens the time for which a preservative is in contact with theprotein, significantly minimizing the associated stability risks. Withliquid formulations, preservative effectiveness and stability should bemaintained over the entire product shelf-life (.about. 18 to 24 months).An important point to note is that preservative effectiveness should bedemonstrated in the final formulation containing the active drug and allexcipient components.

OSMR antigen binding protein formulations generally will be designed forspecific routes and methods of administration, for specificadministration dosages and frequencies of administration, for specifictreatments of specific diseases, with ranges of bio-availability andpersistence, among other things. Formulations thus may be designed inaccordance with the invention for delivery by any suitable route,including but not limited to orally, aurally, opthalmically, rectally,and vaginally, and by parenteral routes, including intravenous andintra-arterial injection, intramuscular injection, and subcutaneousinjection.

Once the pharmaceutical composition has been formulated, it may bestored in sterile vials as a solution, suspension, gel, emulsion, solid,crystal, or as a dehydrated or lyophilized powder. Such formulations maybe stored either in a ready-to-use form or in a form (e.g., lyophilized)that is reconstituted prior to administration. The invention alsoprovides kits for producing a single-dose administration unit. The kitsof the invention may each contain both a first container having a driedprotein and a second container having an aqueous formulation. In certainembodiments of this invention, kits containing single andmulti-chambered pre-filled syringes (e.g., liquid syringes andlyosyringes) are provided.

The therapeutically effective amount of an OSMR antigen bindingprotein-containing pharmaceutical composition to be employed willdepend, for example, upon the therapeutic context and objectives. Oneskilled in the art will appreciate that the appropriate dosage levelsfor treatment will vary depending, in part, upon the molecule delivered,the indication for which the OSMR antigen binding protein is being used,the route of administration, and the size (body weight, body surface ororgan size) and/or condition (the age and general health) of thepatient. In certain embodiments, the clinician may titer the dosage andmodify the route of administration to obtain the optimal therapeuticeffect. A typical dosage may range from about 0.1 μg/kg to up to about30 mg/kg or more, depending on the factors mentioned above. In specificembodiments, the dosage may range from 1.0 μg/kg up to about 20 mg/kg,optionally from 10 μg/kg up to about 10 mg/kg or from 100 μg/kg up toabout 5 mg/kg.

A therapeutic effective amount of an OSMR antigen binding proteinpreferably results in a decrease in severity of disease symptoms, in anincrease in frequency or duration of disease symptom-free periods, or ina prevention of impairment or disability due to the disease affliction.

Pharmaceutical compositions may be administered using a medical device.Examples of medical devices for administering pharmaceuticalcompositions are described in U.S. Pat. Nos. 4,475,196; 4,439,196;4,447,224; 4,447, 233; 4,486,194; 4,487,603; 4,596,556; 4,790,824;4,941,880; 5,064,413; 5,312,335; 5,312,335; 5,383,851; and 5,399,163,all incorporated by reference herein.

Methods of Diagnosing or Treating a OSMR-Associated Disease or Disorder

The OSMR antigen binding proteins of the invention are particularlyuseful for detecting OSMR in a biological sample. In certainembodiments, a biological sample obtained from a patient is contactedwith a OSMR antigen binding protein. Binding of the OSMR antigen bindingprotein to OSMR is then detected to determine the presence or relativeamount of OSMR in the sample. Such methods may be useful in diagnosingor determining patients that are amenable to treatment with an OSMRantigen binding protein.

In certain embodiments, an OSMR antigen binding protein of the inventionis used to diagnose, detect, or treat an autoimmune disorder,inflammatory disorder, or disorder associated with extracellular matrixdeposition or remodeling.

In treating these disorders, the OSMR antigen binding protein may targetOSMR-expressing cells of the immune system for destruction and/or mayblock the interaction of OSMR with OSM and/or IL-31.

Diseases or disorders that are associated with OSMR-mediated signalingare particularly amenable to treatment with one or more OSMR antigenbinding proteins disclosed herein. Such disorders include, but are notlimited to, inflammation, pain, pruritis, prurigo nodularis, dermatitis,asthma, autoimmune disease, paraneoplastic autroimmune diseases,cartilage inflammation, fibrosis (including, but not limited to,pulmonary fibrosis and skin fibrosis), fibrotic disease, chronicobstructive pulmonary disease (COPD), interstitial pneumonitis, abnormalcollagen deposition, systemic cutaneous amyloidosis, primary cutaneousamyloidosis, Behcet's disease, nasal polyposis, liver cirrhosis,cartilage degradation, bone degradation, arthritis, rheumatoidarthritis, juvenile arthritis, juvenile rheumatoid arthritis,pauciarticular juvenile rheumatoid arthritis, polyarticular juvenilerheumatoid arthritis, systemic onset juvenile rheumatoid arthritis,juvenile ankylosing spondylitis, juvenile enteropathic arthritis,juvenile reactive arthritis, juvenile Reter's Syndrome, SEA Syndrome(Seronegativity, Enthesopathy, Arthropathy Syndrome), juveniledermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma,juvenile systemic lupus erythematosus, juvenile vasculitis,pauciarticular rheumatoid arthritis, polyarticular rheumatoid arthritis,systemic onset rheumatoid arthritis, ankylosing spondylitis,enteropathic arthritis, reactive arthritis, Reter's Syndrome, SEASyndrome (Seronegativity, Enthesopathy, Arthropathy Syndrome),dermatomyositis, psoriatic arthritis, scleroderma,scleroderma-associated interstitial lung disease, vasculitis, myolitis,polymyolitis, dermatomyolitis, polyarteritis nodossa, Wegener'sgranulomatosis, arteritis, ploymyalgia rheumatica, sarcoidosis,scleroderma, sclerosis, primary biliary sclerosis, sclerosingcholangitis, Sjogren's syndrome, psoriasis, plaque psoriasis, guttatepsoriasis, inverse psoriasis, pustular psoriasis, erythrodermicpsoriasis, dermatitis, atopic dermatitis, atherosclerosis, lupus,Still's disease, Systemic Lupus Erythematosus (SLE), myasthenia gravis,inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis,celiac disease, multiple sclerosis (MS), asthma, COPD, rhinosinusitis,rhinosinusitis with polyps, eosinophilic esophogitis, eosinophilicbronchitis, bronchitis, Guillain-Barre disease, Type I diabetesmellitus, thyroiditis (e.g., Graves' disease), Addison's disease,Reynaud's phenomenon, autoimmune hepatitis, GVHD, transplantationrejection, kidney damage, cardiovascular disease, infection, sepsis, HIVinfection, trauma, kidney allograft nephropathy, IgA nephropathy,diabetic nephropathy, diabetic retinopathy, macular degeneration,biliary atresia, congestive heart failure, atherosclerosis, restenosis,radiation-induced fibrosis, chemotherapy-induced fibrosis, burns,surgical trauma, glomerulosclerosis, and the like.

In preferred embodiments, the autoimmune disorder, inflammatorydisorder, or disorder associated with extracellular matrix deposition orremodeling is fibrosis, cartilage degradation, arthritis, rheumatoidarthritis, scleroderma, scleroderma-associated interstitial lungdisease, idiopathic pulmonary fibrosis, cirrhosis, psoriasis, atopicdermatitis, systemic cutaneous amyloidosis, primary cutaneousamyloidosis, inflammation, pruritic inflammation, prurigo nodularis, andpain.

In certain embodiments, an OSMR antigen binding protein of the inventionis used to diagnose, detect, or treat a cancer or tumorigenic disorder.In treating a cancer or tumorigenic disorder, the OSMR antigen bindingprotein may target OSMR-expressing cells for destruction and/or mayblock the interaction of OSM and/or IL-31 with OSMR, thereby reducingOSMR mediated signaling. It is contemplated that the OSMR antigenbinding proteins that block OSM- and/or IL-31-mediated signaling wouldbe useful in promoting improved survival in cancer patients. Cancer ortumorigenic disorders that may be diagnosed, detected or treated with anOSMR antigen binding protein include, but are not limited to, solidtumors generally, lung cancer, ovarian cancer, breast cancer, prostatecancer, endometrial cancer, renal cancer, esophageal cancer, pancreaticcancer, squamous cell carcinoma, uveal melanoma, cervical cancer,colorectal cancer, bladder, brain, pancreatic, head, neck, liver,leukemia, lymphoma and Hodgkin's disease, multiple myeloma, melanoma,gastric cancer, astrocytic cancer, stomach, and pulmonaryadenocarcinoma.

The antigen binding proteins may be used to inhibit tumor growth,progression, and/or metastasis. Such inhibition can be monitored usingvarious methods. For instance, inhibition can result in reduced tumorsize and/or a decrease in metabolic activity within a tumor. Both ofthese parameters can be measured by MRI or PET scans, for example.Inhibition can also be monitored by biopsy to ascertain the level ofnecrosis, tumor cell death, and the level of vascularity within thetumor. The extent of metastasis can be monitored using known methods.

The use of any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate embodiments ofthe invention and does not pose a limitation on the scope of theinvention unless otherwise claimed. No language in the specificationshould be construed as indicating any non-claimed element as essentialto the practice of the invention.

EXAMPLES

The following examples, both actual and prophetic, are provided for thepurpose of illustrating specific embodiments or features of the presentinvention and are not intended to limit its scope.

Example 1: Production of Anti-OSMR Antibodies Using the XENOMOUSE®Platform

Fully human antibodies directed against human OSMR were generated usingXENOMOUSE® technology (as described in U.S. Pat. Nos. 6,114,598;6,162,963; 6,833,268; 7,049,426; 7,064,244, which are incorporatedherein by reference in their entirety; and in Green et al., NatureGenetics 7:13-21, 1994; Mendez et al., Nature Genetics 15:146-56; 1997;Green et al., J. Ex. Med. 188:483-95, 1998; and Kellermann et al.,Current Opinion in Biotechnology, 13:593-7, 2002).

To produce antibodies to OSMR, two different strains of XENOMOUSE®animals, i.e., XMG2-KL and XMG4-KL mice, were immunized with humanOSMR-Fc soluble proteins (prepared by Amgen, Seattle, Wash.). A suitableamount of immunogen (i.e., ten μg/mouse of soluble human OSMR-Fcprotein) was used for initial immunization of XENOMOUSE® animalsaccording to the methods disclosed in U.S. patent application Ser. No.08/759,620, filed Dec. 3, 1996 and International Patent Application Nos.WO 98/24893, published Jun. 11, 1998 and WO 00/76310, published Dec. 21,2000, the disclosures of which are hereby incorporated by reference.Following the initial immunization, subsequent boost immunizations ofimmunogen (five μg/mouse of soluble human OSMR-Fc protein) wereadministered on a schedule and for the duration necessary to induce asuitable titer of anti-OSMR antibody in the mice.

Sera were collected at approximately four weeks after the firstinjection and specific titers were determined by ELISA. The protocolused to titer the XENOMOUSE® animals was as follows: Costar 3368 mediumbinding plates were coated with neutravadin @ 8 μg/mL (50 μL/well) andincubated at 4° C. in 1×PBS/0.05% azide overnight. Plates were washedusing TiterTek 3-cycle wash with RO water. Plates were blocked using 250μL of 1×PBS/1% milk and incubated for at least 30 minutes at RT. Blockwas washed off using TiterTek 3-cycle wash with RO water. One thencaptured biotinylated huOSMR-FNFH (prepared by Amgen, Seattle, Wash.) at2 μg/mL in 1×PBS/1% milk/10 mM Ca2+ (assay diluent) 50 μl/well andincubated for 1 hr at RT. One then washed using TiterTek 3-cycle washwith RO water. For the primary antibody, sera was titrated 1:3 induplicate from 1:100. This was done in assay diluent 50 μL/well andincubated for 1 hr at RT. One then washed using TiterTek 3-cycle washwith RO water. The secondary antibody was goat anti Human IgG Fc HRP @400 ng/mL in assay diluent at 50 μL/well. This was incubated for 1 hr atRT. This was then washed using TiterTek 3-cycle wash with RO water andpatted dry on paper towels. For the substrate, one-step TMB solution(Neogen, Lexington, Ky.) was used (50 μL/well) and the substrate wasallowed to develop for 30 min at RT.

Animals exhibiting suitable titers were identified. Five XMG2KL animalswere identified with a specific IgG immune response to OSMR. Spleens anddraining lymph nodes were harvested from these animals and pooledtogether for hybridoma generation. Five XMG4KL animals with specificimmune responses were similarly harvested and advanced as a separatefusion screening campaign. Enriched B cells from immune animals werefused to non-secretory myeloma P3×63Ag8.653 cells ((American TypeCulture Collection CRL-1580; Kearney et al, J. Immunol. 123: 1548-50,1979) to generate hybridomas using standard techniques (Kohler et al.,Nature 256, 495-7, 1975).

Hybridomas were then plated at high density (multiple differenthybridoma clones per well) onto 96-well tissue culture plates and grownfor four weeks. Hybridoma line supernatants were screened for binding tofull length human and cynomolgus OSMR expressed on transientlytransfected 293T cells by Fluorometric Microvolume Assay Technology(FMAT) (Applied Biosystems, Foster City, Calif.). Briefly, in 384-wellFMAT plates, 40 μl mixture of 3,000 OSMR 293T transfected cells and15,000 parental 293T cells were combined with 15 μL of hybridomasupernatant and 10 μL of anti-human light chain (hukappa/hulambda)Alexa647 (Invitrogen, Carlsbad, Calif.) labeled secondary antibody (1.0μg/mL final concentration). Plates were then incubated for three hoursat room temperature and fluorescence was read using the FMAT reader.These screens identified 885 hybridoma lines which bind to both humanand cynomologous OSMR.

Example 2: Human OSMR-Blocking Assays

The ability of OSMR antibodies to block signaling through human OSMR wasdetermined using two assays with either human oncostatin M (OSM) orhuman interleukin 31 (IL-31) as the ligand. In combination, the assayswere used to determine if the antibodies could inhibit signaling of OSMRtriggered through the binding of OSM and/or IL-31.

In the first screen, antibodies were evaluated for their ability toblock the signaling of OSM through OSMR. Stimulation of primary normalhuman lung fibroblasts with OSM induces phosphorylation of STATS and itssubsequent translocation to the nucleus. Cells were seeded at 3000 cellsper well in Costar 384-well plates and allowed to adhere overnight.Cells were pre-treated with antibody supernatants for twenty minutes,and then stimulated with 80 pM human OSM for 30 minutes. Cells were thenwashed 3× in PBS, fixed with a 3.5% formaldehyde solution, washed (3× inPBST) and permeabilized with a 0.5% Triton X-100 solution. Cells werethen stained with an anti-phosphoSTAT3 antibody for an hour, washed andstained with an AlexaFluor conjugated antibody (all contained within theHitKit from Cellomics). Plates were covered and read on the ArrayScaninstrument using the Cellomics proprietary algorithm to generate aNuclear Intensity value and a Cytoplasmic Intensity value. Results werereported as the difference between these two values, and were furthernormalized to control data containing maximally stimulated cells andmedia-treated cells (POC).

In the second assay, antibodies were evaluated for their ability toinhibit a proliferative signal of IL-31 through OSMR in a stable cellline that overexpressed human IL-31RA4 and OSMR. BaF3 cells were stablytransfected with two plasmids: pcDNA3.1+huOSMRb (NeoR) andpcDNA3.1+huIL31RA4 (ZeoR). In the absence of murine 11-3, this cell lineis only able to proliferate in response to human IL-31 and, therefore,could be used to specifically evaluate the blocking ability of anti-OSMRantibodies. BaF3 cells were plated in 96-well plates at a density of20,000 cells per well. Antibodies and ligand (huIL-31, Peprotech) wereadded to the wells to a final volume of 100 μL, and plates wereincubated for 72 hours in 5% CO2, 37 C humidified chamber. Followingincubation, 20 μL of Alamar Blue was added to each well and plates werereturned to the incubator. Plates were read on a Molecular Devices VmaxPlate reader (570-600 nm) at various timepoints post-addition of AlamarBlue.

The results of the two assays are presented in Table 4 below. Over 3000hybridoma supernatants were screened for blocking ability in these twoassays; the top 200 blockers were further tested in a 4-point titration,with 14 being chosen for production of recombinant protein and furthertesting. The IC50 for three exemplary antibodies (antibodies 1-3) isshown for both assays. Some antibodies inhibited OSM-induced STAT3translocation more completely than they inhibited IL-31-inducedproliferation, and vice versa. All three antibodies, however, werepotent inhibitors of OSM- and IL-31 mediated signaling.

TABLE 4 IC50 Ab1 Ab2 Ab3 OSM  157 pM  252 pM 1.35 nM IL-31 35.2 pM 27.6pM  780 pM

Example 3: Cynomolgus OSMR Blocking Assays

The ability of OSMR antibodies to block signaling through cynomolgusOSMR was explored using two assays with either human OSM or human IL-31as the ligand.

In the first screen, antibodies were evaluated for their ability toblock the signaling of OSM through cynomolgus OSMR by using a primarykidney epithelial cell line. Stimulation of these cells with cynomolgus(cyno) OSM induces phosphorylation of STAT3 and its subsequenttranslocation to the nucleus. Cells were seeded at 3000 cells per wellin Costar 384-well plates and allowed to adhere overnight. Cells werepre-treated with antibody supernatants for twenty minutes, and thenstimulated with 80 pM cyno OSM for 30 minutes. Cells were then washed 3×in PBS, fixed with a 3.5% formaldehyde solution, washed (3× in PBST) andpermeabilized with a 0.5% Triton X-100 solution. Cells were then stainedwith an anti-phosphoSTAT3 antibody for an hour, washed and stained withan AlexaFluor conjugated antibody (all contained within the HitKit fromCellomics). Plates were covered and read on the ArrayScan instrumentusing the Cellomics proprietary algorithm to generate a NuclearIntensity value and a Cytoplasmic Intensity value. Results were reportedas the difference between these two values, and were further normalizedto control data containing maximally stimulated cells and media-treatedcells (POC).

In the second assay, antibodies were evaluated for their ability toinhibit a proliferative signal of IL-31 through cynomolgus OSMR in astable cell line that overexpressed cyno IL-31RA and OSMR. Similarly toExample 2, BaF3 cells were plated in 96-well plates at a density of20,000 cells per well. Antibodies and ligand (cynomolgus IL-31,in-house, i.e., Amgen, Seattle, Wash.) were added to the wells to afinal volume of 100 μL, and plates were incubated for 72 hours in 5%CO2, 37 C humidified chamber. Following incubation, 20 μL of Alamar Bluewas added to each well and plates were returned to the incubator. Plateswere read on Molecular Devices Vmax Plate reader (570-600 nm) at varioustimepoints post-addition of Alamar Blue.

The results of the two assays are presented in Table 5 below with theIC50 for each antibody shown for both assays. The results confirm thateach of antibodies 1, 2, and 3 are potent inhibitors of OSM- and IL-31mediated signaling.

TABLE 5 IC50 Ab1 Ab2 Ab3 OSM 1.26 nM  518 pM 1.24 nM IL-31  225 pM 29.3pM 6.87 nM

Example 4: Epitope Binning of Anti-OSMR Antibodies

Antibody competition studies were performed to characterize the epitopesof the anti-OSMR xenomouse antibodies. Antibodies that compete with eachother can be thought of as binding the same site on the target. In theseexperiments, OSMR or irrelevant antibodies were captured ontostreptavidin-coated Luminex beads pre-bound to a capture antibody(biotinylated monovalent mouse anti-human IgG Fc antibody). OSMR antigenor buffer (no antigen) was added to wells, and a probe antibody wasadded to each well and detected with a PE-labeled monovalent mouseanti-human IgG Fc antibody. Mean fluorescence intensity of each well wasmeasured. For a full reference, see Jia et al., J. Immunol. Methods 288:91-8, 2004. Detection of fluorescence in a given well indicated that theprobe antibody was able to bind to OSMR, even in the presence of theother OSMR antibody, demonstrating that they are binding to separateepitopes. A minimum of three bins were found as shown in Table 6 below.

TABLE 6 Bin 1 Ab4 Bin 2 Ab1, Ab2, Ab3, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10,Ab11, Ab12, and Ab13 Bin 3 Ab14

Example 5: Affinity Determination of Anti-OSMR Antibodies

The affinities of anti-OSMR antibodies were determined. Kinetic rateconstant determinations were performed to investigate the interaction ofantibodies 1-3 (Abs 1-3) to human OSMR.

Biosensor analysis was conducted at 25° C. in a HBS-EP+(1×) buffersystem (10 mM HEPES pH 7.4, 150 mM NaCl, 3.0 mM EDTA, 0.05% SurfactantP20) using a Biacore 3000 optical biosensor equipped with a CMS sensorchip. All reagents were kept at 8° C. prior to injection. Goatanti-human IgG (Jackson ImmunoResearch, #109-005-098) was immobilized(˜3000 RU) to the sensor chip via standard amine coupling to Flow Cells1 and 2 and then blocked with ethanolamine hOSMR.FH was prepared inrunning buffer at 150 nM and diluted 3-fold to 0.617 nM. Antibodies 1-3were diluted (0.25 to 0.5 μg/mL) in running buffer. The antibodies wereinjected (15 μL) over Flow Cell 2 at a flow rate of 10 μL/min. About 50RU of antibody was captured. The surface was allowed to stabilize (90 s)and then each concentration (150, 50.0, 16.7, 5.56, 1.85 and 0.617) ofhOSMR was passed over Flow Cells 1 and 2 at a flow rate of 50 μL/min toobserve the association (5 min) and dissociation (5 min). Samples wererun in duplicate and in random order.

Buffer analyte blanks (0 nM hOSMR) were injected before, in-between, andafter sample injections. Antibodies were injected (15 μL) over Flow Cell2 at a flow rate of 10 μL/min. About 50 RU of antibody was captured. Thesurface was allowed to stabilize (90 s) and then each concentration (150nM) of hOSMR was passed over Flow Cells 1 and 2 at a flow rate of 50μL/min to observe the association (5 mM) and dissociation (1-2 hr). Thesamples were run in triplicate.

Buffer analyte blanks (0 nM hOSMR) were injected before and after thesample injections. The surface was regenerated at a flow rate of 50μL/min with two injections of 10 mM glycine (pH 1.5, 50 μL). This wasfollowed by a buffer blank injection (15 s).

Data was analyzed with Scrubber 2.0 software as follows. Data from FlowCell 2 was subtracted from the data from Flow Cell 1 (blank reference).The reference subtracted data (2-1) was then subtracted (doublereferenced) from the nearest 0 nM concentration data. The doublereferenced long dissociation data was fit to a 1:1 binding model todetermine the dissociation rate constant (kd). This dissociation rateconstant was used as a fixed parameter to fit the double referencedshort dissociation data to a 1:1 binding model in order to determine theassociation rate constant (ka) and the equilibrium dissociation constant(Kd).

The reagents were well-behaved under the experimental conditions and thedata (see Table 7 below) fit fairly well to a 1:1 binding model.

TABLE 7 Antibody Antigen k_(a) (1/Ms) k_(d) (1/s) K_(d) (pM) Ab1 huOSMR4.47 × 10⁵ 9.95 × 10⁻⁵ 221 Ab2 huOSMR 5.50 × 10⁵ 1.81 × 10⁻⁵ 32.7 Ab3huOSMR 9.47 × 10⁴ 1.02 × 10⁻⁴ 1080

Example 6: Anti-OSMR Antibodies

Fully human antibodies directed against human OSMR were generated usingXENOMOUSE® technology described above in Example 1. Each of antibodies1, 2, and 3 were demonstrated to be potent inhibitors of OSM- and/orIL-31-mediated signaling. Sequences of antibodies 1, 2, and 3 (i.e.,Ab1, Ab2, and Ab3) were determined and are set forth in Table 8 below.

TABLE 8  SEQ ID Description NO Sequence Ab1- 3caggtgcagctggtgcagtctggggctgaggtgaagaagcctggggc Heavy Chainctcagtgaaggtctcctgcaaggcttctggatacaccttcaccagtt nucleotideatgatatcaactgggtgcgacaggccactggacaggggcttgagtggatgggatggatgaaccctaatagtggtaacacagactatgcacagaagttccagggcagagtcaccatgaccaggaacatttccataagcacggcctacattgagctgagcagcctgagatctgaggacacggccgtttattactgtgcgagagatatggtggctgcgaatacggattactacttctactacggtatggacgtctggggccaagggaccacggtcaccgtctcctcagctagcaccaagggcccatcggtcttccccctggcgccctgctccaggagcacctccgagagcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgctctgaccagcggcgtgcacaccttcccagctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcaacttcggcacccagacctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagacagttgagcgcaaatgttgtgtcgagtgcccaccgtgcccagcaccacctgtggcaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccacgaagaccccgaggtccagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccacgggaggagcagttcaacagcacgttccgtgtggtcagcgtcctcaccgttgtgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccagcccccatcgagaaaaccatctccaaaaccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacacctcccatgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa Ab2- 4caggtgcagctggtgcagtctggggctgaggtgaagaagcctggggc Heavy Chainctcagtgaaggtctcctgcaaggcttctggatacaccttcaccagtt nucleotideatgaaatcaactgggtgcgacaggccactggacaagggcttgagtggatgggatggatgaaccctaacagtggttacacaggctatgcacagaagttccagggcagagtcaccatgaccaggaacacctccataagcacagcctacatggaaatgagcagcctgagatctgaggacacggccgtgtattactgtgcgagagatatagtggctgcgaatacggattactacttctattatggtatggacgtctggggccaagggaccacggtcaccgtctcctcagctagcaccaagggcccatcggtcttccccctggcgccctgctccaggagcacctccgagagcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgctctgaccagcggcgtgcacaccttcccagctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcaacttcggcacccagacctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagacagttgagcgcaaatgttgtgtcgagtgcccaccgtgcccagcaccacctgtggcaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccacgaagaccccgaggtccagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccacgggaggagcagttcaacagcacgttccgtgtggtcagcgtcctcaccgttgtgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccagcccccatcgagaaaaccatctccaaaaccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacacctcccatgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa Ab3- 5caggttcatctggtgcagtctggagctgaggtgaagaagcctggggc Heavy Chainctcagtgaaggtctcctgcaaggcttctggttacacctttaccagct nucleotideatggtatcagctgggtgcgacaggcccctggacaagggcttgagtggatgggatggctcagcacttacagtggtaacacaaactatgcacagaagctccagggcagagtcaccatgaccacagacacatccacgagcacagcctacatggagctgaggagcctgagatctgacgacacggccgtgtattactgtgcgagagggaacttctactactacggtatggacgtctggggccaggggaccacggtcaccgtctcctcagctagcaccaagggcccatcggtcttccccctggcgccctgctccaggagcacctccgagagcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgctctgaccagcggcgtgcacaccttcccagctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcaacttcggcacccagacctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagacagttgagcgcaaatgttgtgtcgagtgcccaccgtgcccagcaccacctgtggcaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccacgaagaccccgaggtccagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccacgggaggagcagttcaacagcacgttccgtgtggtcagcgtcctcaccgttgtgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccagcccccatcgagaaaaccatctccaaaaccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacacctcccatgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcct ctccctgtctccgggtaaa Ab1-6 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDINWVRQATGQGLEW Heavy Chain MGWMNPNSGNTDYAQKFQGRVTMTRNISISTAYIELSSLRSEDTAVY proteinYCARDMVAANTDYYFYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCsRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Ab2- 7QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYEINWVRQATGQGLEW Heavy Chain MGWMNPNSGYTGYAQKFQGRVTMTRNTSISTAYMEMSSLRSEDTAVY proteinYCARDIVAANTDYYFYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Ab3- 8QVHLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEW Heavy Chain MGWLSTYSGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVY proteinYCARGNFYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGKAb1-Heavy 9 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDINWVRQATGQGLEWChain Variable MGWMNPNSGNTDYAQKFQGRVTMTRNISISTAYIELSSLRSEDTAVY RegionYCARDMVAANTDYYFYYGMDVWGQGTTVTVSS Ab2-Heavy  10QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYEINWVRQATGQGLEW Chain VariableMGWMNPNSGYTGYAQKFQGRVTMTRNTSISTAYMEMSSLRSEDTAVY RegionYCARDIVAANTDYYFYYGMDVWGQGTTVTVSS Ab3-Heavy  11QVHLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEW Chain VariableMGWLSTYSGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVY RegionYCARGNFYYYGMDVWGQGTTVTVSS Ab1-Heavy  12 SYDIN Chain CDR1 Ab2-Heavy  13SYEIN Chain CDR1 Ab3-Heavy  14 SYGIS Chain CDR1 Ab1-Heavy  15WMNPNSGNTDYAQKFQG Chain CDR2 Ab2-Heavy  16 WMGWMNPNSGYTGYAQKFQGChain CDR2 Ab3-Heavy  17 WLSTYSGNTNYAQKLQG Chain CDR2 Ab1-Heavy  18DMVAANTDYYFYYGMDV Chain CDR3 Ab2-Heavy  19 DIVAANTDYYFYYGMDV Chain CDR3Ab3-Heavy  20 GNFYYYGMDV Chain CDR3 Ab1- 21cagtctgtgctgactcagccaccctcagcatctgggacccccgggca Light Chain gagggtcaccatctcttgttctggaagcagctccaacgtcggaagta nucleotideatactgtaagctggtaccaacagctcccaggaacggcccccaaactcctcatctatactaataatcggcggccctccggggtccctgaccgattctctggctccaagtctggcacctcagcctccctggccatcagtgggctccagtctgaggatgaggctgattatttctgtgcagcgttagatgacagtctgaatggtgtggtattcggcggagggaccaaactgaccgtcctaggccaaccgaaagcggcgccctcggtcactctgttcccgccctcctctgaggagcttcaagccaacaaggccacactggtgtgtctcataagtgacttctacccgggagccgtgacagtggcctggaaggcagatagcagccccgtcaaggcgggagtggagaccaccacaccctccaaacaaagcaacaacaagtacgcggccagcagctatctgagcctgacgcctgagcagtggaagtcccacagaagctacagctgccaggtcacgcatgaagggagcaccgtggagaagacagtggcccctacagaatgttca Ab2- 22cagtctgtgctgactcagccaccctcagcgtctgggacccccgggca Light Chain gagggtcaccatctcttgttctggaagcaactccaacatcggaagta nucleotideatactgtcaactggtaccaccagctcccaggaacggcccccaaactcctcatctataatattaataagcggccctcaggggtccctgaccgattctctggctccaagtctggctcctcagcctccctggccatcagtgggctccagtctgaggatgaggctgattattactgttcaacatgggatgacagcctggatggtgtggtattcggcggagggaccaagctgaccgtcctaggccaaccgaaagcggcgccctcggtcactctgttcccgccctcctctgaggagcttcaagccaacaaggccacactggtgtgtctcataagtgacttctacccgggagccgtgacagtggcctggaaggcagatagcagccccgtcaaggcgggagtggagaccaccacaccctccaaacaaagcaacaacaagtacgcggccagcagctatctgagcctgacgcctgagcagtggaagtcccacagaagctacagctgccaggtcacgcatgaagggagcaccgtggagaagacagtggcccctacagaatgttca Ab3- 23gaaattgtgttgacgcagtctccaggcaccctgtctttgtctccagg Light Chain ggaaagagccaccctctcctgcagggccagtcagagtgttagcagca nucleotidegctacttagcctggtaccagcagaaacctggccaggctcccaggctcctcatctttggtgcttccagcagggccactggcatcccagacaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagactggagcctgaagattttgcagtgtattactgtcagcagtatggtagctcgcctccgatcaccttcggccaagggacacgactggagattaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt Ab1- 24QSVLTQPPSASGTPGQRVTISCSGSSSNVGSNTVSWYQQLPGTAPKL Light Chain LIYTNNRRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYFCAALDD proteinSLNGVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS Ab2- 25QSVLTQPPSASGTPGQRVTISCSGSNSNIGSNTVNWYHQLPGTAPKL Light Chain LIYNINKRPSGVPDRFSGSKSGSSASLAISGLQSEDEADYYCSTWDD proteinSLDGVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS Ab3- 26EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRL Light Chain LIFGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGS proteinSPPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Ab1- 27QSVLTQPPSASGTPGQRVTISCSGSSSNVGSNTVSWYQQLPGTAPKL Light Chain LIYTNNRRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYFCAALDD VariableSLNGVVFGGGTKLTVLG Ab2- 28QSVLTQPPSASGTPGQRVTISCSGSNSNIGSNTVNWYHQLPGTAPKL Light Chain LIYNINKRPSGVPDRFSGSKSGSSASLAISGLQSEDEADYYCSTWDD VariableSLDGVVFGGGTKLTVLG Ab3- 29EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRL Light Chain LIFGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGS VariableSPPITFGQGTRLEIKR Ab1-Light  30 SGSSSNVGSNTVS Chain CDR1 Ab2-Light  31SGSNSNIGSNTVN Chain CDR1 Ab3-Light  32 RASQSVSSSYLA Chain CDR1Ab1-Light  33 TNNRRPS Chain CDR2 Ab2-Light  34 NINKRPS Chain CDR2Ab3-Light  35 GASSRAT Chain CDR2 Ab1-Light  36 AALDDSLNGVV Chain CDR3Ab2-Light  37 STWDDSLDGVV Chain CDR3 Ab3-Light  38 QQYGSSPPIT Chain CDR3

Example 7: Modified Anti-OSMR Antibodies

Modified versions of Ab1, Ab2, and Ab3 were generated. For all threemodified forms of the antibodies, the lysine at the C terminus of theheavy chain was removed. For Ab1 and Ab2, the glycosylation site atposition 73 was removed by substituting the asparagine at position 73with an aspartic acid. These variants are referred to as Ab1-N73D andAb2-N73D. The sequences of the modified antibodies are set forth inTable 9 below (the modified nucleotides and amino acids are underlined).

TABLE 9  SEQ ID Description NO Sequence Ab1 version 2- 47caggtgcagctggtgcagtctggggctgaggtgaagaagcctggggc Heavy Chainctcagtgaaggtctcctgcaaggcttctggatacaccttcaccagtt nucleotideatgatatcaactgggtgcgacaggccactggacaggggcttgagtgg (N73D/C-terminalatgggatggatgaaccctaatagtggtaacacagactatgcacagaa lysine deleted)gttccagggcagagtcaccatgaccagg g acatttccataagcacggcctacattgagctgagcagcctgagatctgaggacacggccgtttattactgtgcgagagatatggtggctgcgaatacggattactacttctactacggtatggacgtctggggccaagggaccacggtcaccgtctcctcagctagcaccaagggcccatcggtcttccccctggcgccctgctccaggagcacctccgagagcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgctctgaccagcggcgtgcacaccttcccagctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcaacttcggcacccagacctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagacagttgagcgcaaatgttgtgtcgagtgcccaccgtgcccagcaccacctgtggcaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccacgaagaccccgaggtccagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccacgggaggagcagttcaacagcacgttccgtgtggtcagcgtcctcaccgttgtgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccagcccccatcgagaaaaccatctccaaaaccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacacctcccatgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggt Ab2 version 2- 48caggtgcagctggtgcagtctggggctgaggtgaagaagcctggggc Heavy Chainctcagtgaaggtctcctgcaaggcttctggatacaccttcaccagtt nucleotideatgaaatcaactgggtgcgacaggccactggacaagggcttgagtgg (N73D/C-terminalatgggatggatgaaccctaacagtggttacacaggctatgcacagaa lysine deleted)gttccagggcagagtcaccatgaccagg g acacctccataagcacagcctacatggaaatgagcagcctgagatctgaggacacggccgtgtattactgtgcgagagatatagtggctgcgaatacggattactacttctattatggtatggacgtctggggccaagggaccacggtcaccgtctcctcagctagcaccaagggcccatcggtcttccccctggcgccctgctccaggagcacctccgagagcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgctctgaccagcggcgtgcacaccttcccagctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcaacttcggcacccagacctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagacagttgagcgcaaatgttgtgtcgagtgcccaccgtgcccagcaccacctgtggcaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccacgaagaccccgaggtccagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccacgggaggagcagttcaacagcacgttccgtgtggtcagcgtcctcaccgttgtgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccagcccccatcgagaaaaccatctccaaaaccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacacctcccatgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggt Ab3- 49caggttcatctggtgcagtctggagctgaggtgaagaagcctggggc Heavy Chainctcagtgaaggtctcctgcaaggcttctggttacacctttaccagct nucleotideatggtatcagctgggtgcgacaggcccctggacaagggcttgagtgg (C-terminal lysineatgggatggctcagcacttacagtggtaacacaaactatgcacagaa deleted)gctccagggcagagtcaccatgaccacagacacatccacgagcacagcctacatggagctgaggagcctgagatctgacgacacggccgtgtattactgtgcgagagggaacttctactactacggtatggacgtctggggccaggggaccacggtcaccgtctcctcagctagcaccaagggcccatcggtcttccccctggcgccctgctccaggagcacctccgagagcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgctctgaccagcggcgtgcacaccttcccagctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcaacttcggcacccagacctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagacagttgagcgcaaatgttgtgtcgagtgcccaccgtgcccagcaccacctgtggcaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccacgaagaccccgaggtccagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccacgggaggagcagttcaacagcacgttccgtgtggtcagcgtcctcaccgttgtgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccagcccccatcgagaaaaccatctccaaaaccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacacctcccatgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcct ctccctgtctccgggtAb1 version 2- 50 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDINWVRQATGQGLEWHeavy Chain protein MGWMNPNSGNTDYAQKFQGRVTMTR D ISISTAYIELSSLRSEDTAVY(N73D/C-terminal YCARDMVAANTDYYFYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSlysine deleted) RSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Ab2 version 2- 51QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYEINWVRQATGQGLEW Heavy Chain proteinMGWMNPNSGYTGYAQKFQGRVTMTR D TSISTAYMEMSSLRSEDTAVY (N73D/C-terminalYCARDIVAANTDYYFYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCS lysine deleted)RSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Ab3 version 2- 52QVHLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEW Heavy Chain proteinMGWLSTYSGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVY (C-terminal lysineYCARGNFYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSEST deleted)AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGAb1 version 2- 53 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDINWVRQATGQGLEWHeavy Chain Variable MGWMNPNSGNTDYAQKFQGRVTMTR D ISISTAYIELSSLRSEDTAVYRegion YCARDMVAANTDYYFYYGMDVWGQGTTVTVSS (N73D/C-terminal lysine deleted)Ab2 version 2- 54 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYEINWVRQATGQGLEWHeavy Chain Variable MGWMNPNSGYTGYAQKFQGRVTMTR D TSISTAYMEMSSLRSEDTAVYRegion YCARDIVAANTDYYFYYGMDVWGQGTTVTVSS (N73D/C-terminal lysine deleted)

ELISA experiments were performed under various formats (Capture ELISAfor avidity-less format; Sandwich ELISA for solution phase format; andDirect ELISA for solid state avidity format) using antibodies containingthe variable regions of Ab1 or Ab2 (or the N73D variant of Ab1 or Ab2)with different Fc regions.

Ab1 and Ab2 each contain CH1, CH2, CH3 domains of human IgG2 origin. Asused herein, the terms “Ab1” and “Ab1 IgG2 WT” refer to the sameantibody. Similarly, the terms “Ab2” and “Ab2 IgG2 WT” refer to the sameantibody.

Antibodies identified as “IgG4P agly/IgG1” contain the variable regionsof Ab1 or Ab2 (or the N73D variant of Ab1 or Ab2) fused to the CH1domain from human IgG4, the hinge from human IgG4 with a Ser to Promutation (at position 228) to reduce shuffling, the CH2 domain fromhuman IgG4 with an Asn to Gln mutation (at position 297) to eliminatethe N-linked glycosylation site, and the CH3 domain from human IgG1. The“IgG4P agly/IgG1” framework is described in US published patentapplication number US 2012/0100140.

The ELISA results indicated that removal of glycosylation sites via theN73D substitution did not affect the binding of the modified antibodiesto OSMR. See Table 10.

TABLE 10 Capture (EC50) Sandwich (EC50) Direct (EC50) Antibody nM nM nMAb1 IgG2 WT 10.2 0.581 0.184 Ab1 N73D IgG2 4.85 0.359 0.0728 Ab1 N73DIgG4P 2.86 0.05 0.0626 agly/IgG1 Ab2 IgG2 WT 3.63 0.366 0.182 Ab2 N73DIgG2 5.49 0.343 0.179 Ab2 N73D IgG4P 1.61 0.064 0.0558 agly/IgG1

Binding studies were performed using the BIAcore method. Antibodiescontaining the variable regions of Ab1 or Ab2 (or the N73D variant ofAb1 or Ab2) with different Fc regions were immobilized on a CM4 chip (GElifesciences) as per manufacturer's protocols. Soluble OSMR was used asthe analyte. Removing the glycosylation site on Ab1 and Ab2 via the N73Dsubstitution improved the binding affinities. For Ab1, the substitutionimproved Kon rate, whereas for Ab2 it improved Koff rate. See Table 11.

TABLE 11 Antibody Kon (M−1s−1) Koff (1/s) KD (M) Ab1 IgG2 WT 1.64E+051.50E−04 0.913E−9 Ab1 N73D IgG2 2.49E+05 1.68E−04 0.675E−9 Ab2 IgG2 WT1.88E+05 1.89E−04  1.01E−09 Ab2 N73D IgG2 1.73E+05 4.99E−05 0.289E−9

The stability of Fab fragments was determined by assessing the thermalunfolding of antibodies. High melting temperature of Fab fragmentscorrelates directly to increased stability. Removal of glycosylationsites on Ab2 via the N73D substitution did not affect the thermalstability of Fab fragments and showed minor effects on Ab1, as assessedby differential scanning fluorimery experiments. See Table 12.

TABLE 12 Antibody Fab Tm (Celsius) Standard Error (Celsius) Ab1 IgG2 WT73.24 0.0097 Ab1 N73D IgG2 71.21 0.005 Ab1 N73D IgG4P 74.23 0.014agly/IgG1 Ab2 IgG2 WT 76.71 0.0096 Ab2 N73D IgG2 76.54 0.14 Ab2 N73DIgG4P 76.69 0.018 agly/IgG1

The ability of modified anti-OSMR antibodies to block signaling throughhuman OSMR was assessed. The modified antibodies were evaluated fortheir ability to inhibit proliferation of a BaF_hu-IL31R/OSMR/gp130 cellline in the presence of IL31, OSM, or IL31 and OSM. The results arepresented in Tables 13 and 14 below, with the IC50 for each antibodyshown. The results confirm that the modified versions of Ab1 and Ab2 arepotent inhibitors of OSM- and IL-31-mediated signaling.

TABLE 13 IL31 OSM IL31/OSM Antibody (IC50) (IC50) (IC50) Ab2 0.38280.3528 ~1.9 Ab1 IgG2 WT 0.6691 0.5298 4.004 Ab1 N73D IgG2 0.7565 0.52263.702 Ab1 N73D IgG4P 0.4672 0.5657 3.080 agly/IgG1

TABLE 14 IL31 OSM IL31/OSM Antibody (IC50) (IC50) (IC50) Ab2 0.44260.4019 1.8 Ab2 IgG2 WT 0.3671 0.4758 2.049 Ab2 N73D IgG2 0.2191 0.18591.474 Ab2 N73D IgG4P 0.2838 0.2401 1.276 agly/IgG1

The disclosure has been described in terms of particular embodimentsfound or proposed to comprise specific modes for the practice of thedisclosure. Various modifications and variations of the describedinvention will be apparent to those skilled in the art without departingfrom the scope and spirit of the invention. Although the invention hasbeen described in connection with specific embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention that are obvious to thoseskilled in the relevant fields are intended to be within the scope ofthe following claims.

1-74. (canceled)
 75. A pharmaceutical composition comprising ananti-oncostatin M receptor (OSMR) antibody and a carrier, wherein theanti-OSMR antibody comprises: a light chain variable domain comprising alight chain complementary-determining region 1 (LCDR1) defined by SEQ IDNO:31, a light chain complementary-determining region 2 (LCDR2) definedby SEQ ID NO:34, and a light chain complementary-determining region 3(LCDR3) defined by SEQ ID NO:37; and a heavy chain variable domaincomprising a heavy chain complementary-determining region 1 (HCDR1)defined by SEQ ID NO:13, a heavy chain complementary-determining region2 (HCDR2) defined by SEQ ID NO:16, and a heavy chaincomplementary-determining region 3 (HCDR3) defined by SEQ ID NO:19. 76.The pharmaceutical composition of claim 75, wherein the light chainvariable domain has the amino acid sequence set forth in SEQ ID NO:28.77. The pharmaceutical composition of claim 75, wherein the heavy chainvariable domain has the amino acid sequence set forth in SEQ ID NO:54.78. The pharmaceutical composition of claim 75, wherein: the light chainvariable domain has the amino acid sequence set forth in SEQ ID NO:28;and the heavy chain variable domain has the amino acid sequence setforth in SEQ ID NO:54.
 79. The pharmaceutical composition of claim 75,wherein the light chain of the antibody comprises the amino acidsequence set forth in SEQ ID NO:25.
 80. (canceled)
 81. Thepharmaceutical composition of claim 75, wherein the antibody is a humanantibody.
 82. The pharmaceutical composition of claim 75, wherein theantibody inhibits binding of human oncostatin M (OSM) or humaninterleukin 31 to human OSMR.
 83. The pharmaceutical composition ofclaim 75, wherein the antibody reduces human OSM-mediated or humaninterleukin 31-mediated OSMR signaling in human OSMR-expressing cells.84. (canceled)
 85. The pharmaceutical composition of claim 78, whereinthe antibody is a human antibody.
 86. The pharmaceutical composition ofclaim 78, wherein the light chain of the antibody comprises the aminoacid sequence set forth in SEQ ID NO:25.
 87. The pharmaceuticalcomposition of claim 78, wherein the antibody inhibits binding of humanOSM or human interleukin 31 to human OSMR.
 88. The pharmaceuticalcomposition of claim 78, wherein the antibody reduces human OSM-mediatedor human interleukin 31-mediated OSMR signaling in human OSMR-expressingcells.
 89. The pharmaceutical composition of claim 75, wherein thecarrier is aqueous.
 90. The pharmaceutical composition of claim 75,wherein the carrier is non-aqueous.
 91. The pharmaceutical compositionof claim 75, wherein the pharmaceutical composition is a solution. 92.The pharmaceutical composition of claim 75, wherein the pharmaceuticalcomposition is lyophilized.